Modified immunization vectors

ABSTRACT

The disclosure relates to recombinant vectors and methods for using the same. In certain embodiments, the recombinant vectors are immunogenic.

PRIOR APPLICATIONS

This application is a continuation application of U.S. Ser. No.15/289,297 having a filing date of Oct. 10, 2016, which is acontinuation application of U.S. Ser. No. 13/266,282 having a filingdate of Jan. 6, 2012, which was filed under 35 U.S.C. section 371, andclaims priority to International Application No PCT/US2010/032966 filedApr. 29, 2010, and claims priority to U.S. Ser. No. 61/174,024 filedApr. 30, 2009.

FIELD OF THE INVENTION

The disclosure relates to modified vectors for use in immunologicalcompositions.

BACKGROUND OF THE INVENTION

There is need in the art for effective immunological compositions andmethods for immunizing animals and humans using recombinant vectors. Itis known in the art that certain vectors (e.g., replication-incompetentvaccinia vectors) are insufficient as immunomodulators. As describedherein, modification of such vectors provides a solution to theseproblems.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Plasmid maps of transfer vectors.

FIG. 2. Schematic representation of NYVAC genome.

FIG. 3A. Constuction of the plasmid transfer vector pGem-RG-B8R wm.

FIG. 3B. pGem-RG-B8R wm plot.

FIG. 4. PCR analysis of NYVAC-C-ΔB8R, NYVAC-C-ΔB19R andNYVAC-C-ΔB8RB19R.

FIG. 5. Immunoblot analysis of NYVAC-C-ΔB8R, NYVAC-C-ΔB19R andNYVAC-C-ΔB8RB19R.

FIG. 6. Immunostain analysis of NYVAC-C-ΔB8R, NYVAC-C-ΔB19R andNYVAC-C-ΔB8RB19R.

FIG. 7. Virus growth curves of NYVAC-C-ΔB8R, NYVAC-C-ΔB19R andNYVAC-C-ΔB8RB19R.

FIG. 8A. Construction of plasmid transfer vector pGem-RG-B19R wm.

FIG. 8B. pGem-RG-B19R wm plot.

FIG. 9. Methodology used to construct KC viruses. To create a singlefragment containing K1L and C7L, the two genes were first amplified byPCR from the wild type vaccinia virus genome, Copenhagen strain. PCR wasused to fuse the two fragments into one. In vivo recombination (IVR) wasused to insert the final PCR product between the existing inter-genicregions of the genome, creating NYVAC. In vivo combination was also usedto create NYVAC-C-KC-ΔB8R-ΔB19R, using NYVAC-C-ΔB8R-ΔB19R as theparental virus.

FIG. 10. Methodology used to construct NYVAC-C+12 virus.

FIG. 11. Methdology used to construct NYVAC-C+12-ATVh virus. The E3Lgene of NYVAC was replaced with the eIF2a homologue from Ambystomatigrinum virus (ATVh) by in vivo recombination (IVR) to createNYVAC-C+12-ATVh. The ATVh had been amplified by PCR, with flankingregion sequences at either end of the gene to allow for recombination,and inserted into a transfer plasmid. The plasmid was used in the IVR totransfer the ATVh into the virus genome.

FIGS. 12A and 12B. Flow cytometric analysis.

FIG. 13A. Upregulation of costimulatory molecules on infected humanmoDCs. IL-4 and GM-CSF differentiated DC were infected with NYVAC-C andthe deletion mutants B19R (A) and B8R/B19R (B) expression ofcostimulatory molecules was analyzed by FACS analysis 48 hr postinfection. DCs were infected with an MOI of 0.1. The shaded peaks in thehistograms represent NYVAC-wt infected DC; the unshaded peaks representDC infected with NYVAC-C, B19R or B8R/B19R.

FIG. 13B. Cytokine production by HIV specific CD8 T cells in a directand cross presentation assay. DCs were infected or incubated withapoptotic infected HeLa cells for 6 hrs before CD8 T cells were added.After overnight incubation the amount of single, double and triplecytokine producing cells was determined by FACS analysis.

FIG. 14A. IL-8 production assays by human THP-1 macrophages infectedwith wild-type and mutant NYVAC and NYVAC-C.

FIG. 14B. IL-8 production assays by human THP-1 macrophages infectedwith wild-type and mutant NYVAC and NYVAC-C.

FIG. 14C. IL-8 production assays and whole blood infected with wild-typeand mutant NYVAC and NYVAC-C.

FIG. 14D. IL-8 production assays and whole blood infected with wild-typeand mutant NYVAC and NYVAC-C.

FIG. 15A. Chemokine and cytokine expression levels.

FIG. 15B. IFN expression levels.

FIG. 15C. Enhanced expression of pathogen sensing molecules.

FIG. 15D. Enhanced expression in genes associated with inflammatoryresponse.

SUMMARY OF THE DISCLOSURE

Disclosed herein are compositions and reagents for immunizing humanbeings against infectious or other agents such as tumor cells byinducing or enhancing thereto. In certain embodiments, the compositionscomprise recombinant viral vectors comprising modified nucleotidesequences. In certain embodiments, the vectors were modified by deletionof and/or insertion of nucleic acids encoding any one or more of thepolypeptides shown in SEQ ID NOS. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19. 21.23, 25, or 27. Exemplary of such polynucleotides are those shown in SEQID NOS. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, and 28. In someembodiments, such vectors further comprise polynucleotides encodingimmunogens. Methods for constructing and using such vectors aredescribed herein. Compositions comprising such vectors and methods forusing such compostions are also provided.

DETAILED DESCRIPTION

The present disclosure provides compositions and methodologies usefulfor expressing nucleic acids and the polypeptides, peptides, or nucleicacids encoded thereby using recombinant vectors. In one embodiment, thecompositions comprise recombinant vectors for introducing or alteringthe expression of a polypeptide, peptide, or nucleic acid in a host. Insome embodiments, the compositions may include one or more recombinantviruses comprising polynucleotides encoding polypeptides, peptides, orpolynucleotides that were not previously expressed by the virus, or arenormally expressed in different amounts or at different times in thelife cycle of the virus. In certain embodiments, polynucleotides areincorporated into the genome of a virus to produce a recombinant viruswith altered characteristics as compared to the non-modified virus. Insome embodiments, the incorporated polynucleotides encode polypeptides,peptides, or polynucleotides that alter the growth characteristics,infectivity, host range, replicative capacity, or immunogenicity of therecombinant virus as compared to the non-modified virus. Suchpolynucleotides may be used alone or in combination with otherpolynucleotides such as those described below (e.g., encoding one ormore immunogens).

Expression vectors may also be modified by deleting polynucleotides(e.g., a gene) normally found within the vector therefrom. For instance,the poxvirus NYVAC (described in more detail below) was derived from theCopenhagen vaccinia strain using transient dominant selection (Falkner &Moss, 1990) which allows for deletion of one or more target geneswithout incorporation of a polynucleotide encoding a selectable markerinto the viral genome. Polynucleotides may be completely or partiallydeleted, or inactivated with or without partial deletion. Partialdeletion may be accomplished by removing a portion of a polynucleotideencoding a polypeptide from the “genome” of the vector (“vectorgenome”). As referred to herein, the vector genome may refer to thepolynucleotide encoding the various factors required for the viabilityof a replication-competent or replication-incompetent viral vector, thepolynucleotide making up a non-viral (e.g., bacterial, eukaryotic) orviral plasmid vector, or the like.

For instance, NYVAC is derived from the VACV strain Copenhagen (COP)from which 18 genes encoding proteins involved in host range andvirulence were deleted (Tartaglia et al., 1992). These vectors wereshown to exhibit altered host range and to be useful for expressingimmunogens within a wide range of species (Tartaglia et al., 1994). Suchvectors have been used as recombinant vaccines against numerouspathogens and tumours in animal models and in target species, includinghumans (Myagkikh et al., 1996; Benson et al., 1998; Siemens et al.,2003; Franchini et al., 2004). Clinical trials using NYVAC-based vectorsshowed an acceptable safety profile, with induction of high levels ofimmunity against heterologous antigens (Kanesa-thasan et al., 2000;Gómez, C. E el al. 2007; Harari, A et al, 2008). Such vectors may befurther modified by insertion or deletion of additional polynucleotidesusing the techniques described herein. Suitable polynucleotides mayinclude, for example, those involved in host range, apoptosis,signaling, cytokine and/or chemokine expression or activity, cytokineand/or chemokine pathways, and/or the like, resulting in novelbiological characteristics of the vectors.

In some embodiments, polynucleotides encoding immunomodulatorypolypeptides are selectively deleted from a vector genome.Polynucleotides encoding immunogens may also be incorporated into thevector genome. This may lead to modulation of virus-host cellinteractions and “improvement” in the immunological profiles of themodified vectors as candidate vaccines. By “improvement” is meant thatan immune response against a target antigen is induced or enhanced. Incertain embodiments, the modified vectors may exhibit improved safetyprofiles as compared to non-modified (e.g., parental) vectors.

Polynucleotides suitable to modification (e.g., deletion from,alteration of sequence, or incorporation into a vector genome) mayinclude, for example, any polynucleotide that provides the desiredeffect (e.g., an improved immune response). For instance, within theNYVAC vector, candidate polynucleotides may include polynucleotides thatmay be characterized as immunomodulators, and those affecting viral hostrange, one or more signalling pathways, apoptosis, secreted proteins(e.g., those binding host cytokines and/or chemokines). Exemplarypolynucleotides and polypeptides that are candidates for modificationinclude those encoding, for example, B8R (SEQ ID NOS. 1, 2) and/or B19R(SEQ ID NOS. 3, 4). In certain embodiments, suitable and exemplarypolynucleotides may encode immunomodulatory polypeptides that interactwith, for example, one or more interferons, cytokines and/or chemokines(e.g., B8R (SEQ ID NOS. 1, 2), and/or B19R (SEQ ID NOS. 3, 4)). Thenomenclature of these sequences is related to the Copenhagen strain ofvaccinia virus (GenBank Accession No. M35027; Goebel, et al. Thecomplete DNA sequence of vaccinia virus. Virology 179 (1), 247-266(1990); Goebel, et al. Appendix to ‘The complete DNA sequence ofVaccinia virus’. Virology 179, 517-563 (1990)). Any of suchpolynucleotides may be modified (e.g, incorporated into a recombinantvector or as part of a composition containing multiple recombinantvectors) in combination with any other of such polynucleotides. Otherpolynucleotides may also be suitable for modification in vaccinia or inother viruses (e.g., MVA, avipox, and the like).

The B8R gene (open reading frame (“ORF”) shown in SEQ ID NO. 1) encodesthe B8R protein (SEQ ID NO. 2) with amino acid similarity to theextracellular domain of the IFN-γ receptor (Alcami & Smith, 1995;Mossman et al., 1995). The protein B8 binds and inhibits IFN-γ from awide variety of species but not the mouse. Deletion of B8R from WR didnot alter virus replication or virulence in mouse models (Symons, et al.2002a).

The B19R gene of VACV (ORF shown in SEQ ID NO. 3 encoding the B19Rpolypeptide, SEQ ID NO. 4) is equivalent to the B18R gene of VACV WR andencodes a type I IFN (α,β)-receptor homolog. Protein B19 binds andinhibits type I IFN from a wide variety of species except murine IFNwhich binds but does not inhibit it. Deletion of B19R from VACV WR hasbeen shown to cause attenuation in a murine intranasal model.

Within vaccinia, the C1L (SEQ ID NO. 5), C2L (SEQ ID NO. 7), C3L (SEQ IDNO. 9), C4L (SEQ ID NO. 11), C5L (SEQ ID NO. 13), C6L (SEQ ID NO. 15),C7L (SEQ ID NO. 17), N1L (SEQ ID NO. 19), N2L (SEQ ID NO. 21), M1L (SEQID NO. 23), M2L (SEQ ID NO. 25) and K1L (SEQ ID NO. 27) polypeptideshave been shown to be involved in defining the “host range” orreplication competence of the virus. Polynucleotides encoding such hostrange polypeptides are illustrated in SEQ ID NOS. 6, 8, 10, 12, 14, 16,18, 20, 22, 24, 26, and 28. In the NYVAC virus, these genes have beendeleted. In certain embodiments, one or more polynucleotidesrepresenting one or more of these host range genes may be introducedinto the genome of a viral vector to affect the replication competenceof the vector. In NYVAC, for example, one or more polynucleotidesrepresenting one or more of such host range genes may be re-incorporatedinto the NYVAC genome to modify its replication competence. In certainembodiments, as shown in the Examples, polynucleotides encoding C7L(e.g., SEQ ID NOS. 17, 18) and K1L (e.g., SEQ ID NOS. 27, 28) were shownto effect replication competence of NYVAC. In certain embodiments, therecombinant vector (e.g., NYVAC) expresses at least one, two, three,four, five, six, seven, eight, nine, ten, eleven or twelve of C1L (e.g.,SEQ ID NOS. 5, 6), C2L (e.g., SEQ ID NOS. 7, 8), C3L (e.g., SEQ ID NOS.9, 10), C4L (SEQ ID NOS. 11, 12), C5L (e.g., SEQ ID NOS. 13, 14), C6L(e.g., SEQ ID NOS. 15, 16), C7L (e.g., SEQ ID NOS. 17, 18), N1L (SEQ IDNOS. 19, 20), N2L (e.g., SEQ ID NOS. 21, 22), M1L (e.g., SEQ ID NOS. 23,24), M2L (e.g., SEQ ID NOS. 25, 26), and K1L (e.g., SEQ ID NOS. 27, 28).Various combinations of such polynucleotides and/or polypeptides, aswould be apparent to one of skill in the art, may be utilized invectors. These polynucleotides and/or polypeptides may also beincorporated into vectors engineered to contain or express otherpolynucleotides and/or polypeptides such as, for example, B8R (SEQ IDNOS. 1, 2) and/or B19R (SEQ ID NOS. 3, 4). Suitable recombinant vectorsfor introduction or re-introduction of such host range genes includethose from which such sequences have been previously deleted or thosethat otherwise do not contain such genes within the vector genome. It isalso possible to modify such host range genes such that their functionis altered by, for example, altering the timing or character (e.g.,expression level) of expression within a host cell.

Polynucleotides encoding other polypeptides, peptides, or nucleic acidsaffecting the activity of a recombinant vector (e.g., recombinant virus)may also be incorporated into the vector. In certain embodiments,polynucleotides representing genes from other organisms (exogenousgenes) may be incorporated into the vector. The polynucleotides may beinserted into a polynucleotide by insertion, either de novo or byreplacement of an existing polynucleotide sequence within the vectorgenome. For instance, a polynucleotide may replace a gene of a virus.For example, the ranavirus eIF2α-like gene (“eIF2αH”) from Ambystomatigrinum virus isolate YEL protein gene (GenBank Accession No. EU512333;version EU512333.1; GI:170180537; “ATV eIF2αH”; SEQ ID NO. 29 encoded bySEQ ID NO. 30; see, e.g., U.S. Pat. No. 7,431,929) may be utilized. ATVeIF2αH encodes a potent, non-dsRNA-binding inhibitor of RNA-dependentprotein kinase (PKR). In one embodiment, a polynucleotide encoding ATVeIF2αH (e.g., SEQ ID NO. 30) may be incorporated into a recombinantvector described herein. Without being limited to any particular theoryof operation, it is believed that ATV eIF2αH induces signal transductionthrough NF-κB and IRF-3, while sparing viral protein synthesis from theinhibitory effects of PKR activation. In certain embodiments, arecombinant virus may be produced that exhibits little, decreased, or noreplication competence but also induces an immune response in a host.Such a virus may provide an optimal recombinant vector that represents a“compromise” between replication competent that may cause complicationsin hosts, and replication deficient recombinant vectors that may fail toinduce an immune response, or may induce a sub-optimal immune response.

In certain embodiments, in addition to the one or more polynucleotidesencoding one or more of B8R (SEQ ID NOS. 1, 2) and/or B19R (SEQ ID NOS.3, 4)), the recombinant vector may also comprise a polynucleotideencoding ATV eIF2αH (e.g., SEQ ID NOS. 29, 30) such as SEQ ID NO. 54. Inother embodiments, a recombinant vector including any one or more of C1L(e.g., SEQ ID NOS. 5, 6), C2L (e.g., SEQ ID NOS. 7, 8), C3L (e.g., SEQID NOS. 9, 10), C4L (SEQ ID NOS. 11, 12), C5L (e.g., SEQ ID NOS. 13,14), C6L (e.g., SEQ ID NOS. 15, 16), C7L (e.g., SEQ ID NOS. 17, 18), N1L(SEQ ID NOS. 19, 20), N2L (e.g., SEQ ID NOS. 21, 22), M1L (e.g., SEQ IDNOS. 23, 24), M2L (e.g., SEQ ID NOS. 25, 26), and/or K1L (e.g., SEQ IDNOS. 27, 28) (or a deletion of any one or more of these sequences) mayalso comprise a polynucleotide encoding ATV eIF2αH (e.g., SEQ ID NOS.29, 30). In yet other embodiments, a recombinant vector may alsocomprise one or more polynucleotides encoding one or more of B8R (SEQ IDNOS. 1, 2) and/or B19R (SEQ ID NOS. 3, 4)) and/or a polynucleotideencoding ATV eIF2αH (e.g., SEQ ID NOS. 29, 30), and/or any one or moreof C1L (e.g., SEQ ID NOS. 5, 6), C2L (e.g., SEQ ID NOS. 7, 8), C3L(e.g., SEQ ID NOS. 9, 10), C4L (SEQ ID NOS. 11, 12), C5L (e.g., SEQ IDNOS. 13, 14), C6L (e.g., SEQ ID NOS. 15, 16), C7L (e.g., SEQ ID NOS. 17,18), N1L (SEQ ID NOS. 19, 20), N2L (e.g., SEQ ID NOS. 21, 22), M1L(e.g., SEQ ID NOS. 23, 24), M2L (e.g., SEQ ID NOS. 25, 26), and/or K1L(e.g., SEQ ID NOS. 27, 28). For instance, the Examples demonstrate arecombinant vaccinia virus in which the E3L gene was deleted andreplaced by a polynucleotide encoding ATV eIF2αH (SEQ ID NO. 30 encodingSEQ ID NO. 29; see, e.g., U.S. Pat. No. 7,431,929). It was observed thatthis modified virus induces host cell production of IFN, exhibitsincreased sensitivity to IFN, and induces a potent Th1-dominated immuneresponse at low doses. Other embodiments, as could be derived from thisdisclosure, may also be suitable for use.

In some embodiments, the compositions may include one or morerecombinant vectors encoding one or more immunogens that may be used toinduce or enhance an immune response that is beneficial to the host. Assuch, the compositions described herein may also be used to treat and/orprevent conditions relating to an infectious or other agent(s) byinducing or enhancing an immune response against such an agent. Incertain embodiments, the compositions may comprise one or morerecombinant vectors encoding one or more immunogens (e.g., comprising apolynucleotide encoding the antigen). An immunogen may be isolated fromits source (e.g., an infectious agent) of which it forms a part (e.g., apolypeptide normally found within or expressed by that infectiousagent). In certain embodiments, the immunogen may be encoded by anucleotide sequence in expressible form (e.g., within an expressionvector).

An immunogen may be a moiety (e.g., polypeptide, peptide or nucleicacid) that induces or enhances the immune response of a host to whom orto which the immunogen is administered. An immune response may beinduced or enhanced by either increasing or decreasing the frequency,amount, or half-life of a particular immune modulator (e.g, theexpression of a cytokine, chemokine, co-stimulatory molecule). This maybe directly observed within a host cell containing a polynucleotide ofinterest (e.g., following infection by a recombinant virus) or within anearby cell or tissue (e.g., indirectly). The immune response istypically directed against a target antigen. For example, an immuneresponse may result from expression of an immunogen in a host followingadministration of a nucleic acid vector encoding the immunogen to thehost. The immune response may result in one or more of an effect (e.g.,maturation, proliferation, direct- or cross-presentation of antigen,gene expression profile) on cells of either the innate or adaptiveimmune system. For example, the immune response may involve, effect, orbe detected in innate immune cells such as, for example, dendriticcells, monocytes, macrophages, natural killer cells, and/or granulocytes(e.g., neutrophils, basophils or eosinophils). The immune response mayalso involve, effect, or be detected in adaptive immune cells including,for example, lymphocytes (e.g., T cells and/or B cells). The immuneresponse may be observed by detecting such involvement or effectsincluding, for example, the presence, absence, or altered (e.g.,increased or decreased) expression or activity of one or moreimmunomodulators such as a hormone, cytokine, interleukin (e.g., any ofIL-1 through IL-35), interferon (e.g., any of IFN-I (IFN-α, IFN-β,IFN-ε, IFN-κ, IFN-τ, IFN-ζ, IFN-ω), IFN-II (e.g., IFN-γ), IFN-III(IFN-λ1, IFN-λ2, IFN-λ3)), chemokine (e.g., any CC cytokine (e.g., anyof CCL1 through CCL28), any CXC chemokine (e.g., any of CXCL1 throughCXCL24), Mip1a), any C chemokine (e.g., XCL1, XCL2), any CX3C chemokine(e.g., CX3CL1)), tumor necrosis factor (e.g., TNF-α, TNF-β)), negativeregulators (e.g., PD-1, IL-T) and/or any of the cellular components(e.g., kinases, lipases, nucleases, transcription-related factors (e.g.,IRF-1, IRF-7, STAT-5, NFKB, STAT3, STAT1, IRF-10), and/or cell surfacemarkers suppressed or induced by such immunomodulators) involved in theexpression of such immunomodulators. The presence, absence or alteredexpression may be detected within cells of interest or near those cells(e.g., within a cell culture supernatant, nearby cell or tissue in vitroor in vivo, and/or in blood or plasma). Administration of the immunogenmay induce (e.g., stimulate a de novo or previously undetectedresponse), or enhance or suppress an existing response against theimmunogen by, for example, causing an increased antibody response (e.g.,amount of antibody, increased affinity/avidity) or an increased cellularresponse (e.g., increased number of activated T cells, increasedaffinity/avidity of T cell receptors). In certain embodiments, theimmune response may be protective, meaning that the immune response maybe capable of preventing initiation or continued infection of or growthwithin a host and/or by eliminating an agent (e.g., a causative agent,such as HIV) from the host.

The compositions described herein may include one or more immunogen(s)from a single source or multiple sources. For instance, immunogens mayalso be derived from or direct an immune response against one or moreviruses (e.g., viral target antigen(s)) including, for example, a dsDNAvirus (e.g. adenovirus, herpesvirus, epstein-barr virus, herpes simplextype 1, herpes simplex type 2, human herpes virus simplex type 8, humancytomegalovirus, varicella-zoster virus, poxvirus); ssDNA virus (e.g.,parvovirus, papillomavirus (e.g., E1, E2, E3, E4, E5, E6, E7, E8, BPV1,BPV2, BPV3, BPV4, BPV5 and BPV6 (In Papillomavirus and Human Cancer,edited by H. Pfister (CRC Press, Inc. 1990); Lancaster et al., CancerMetast. Rev. pp. 6653-6664 (1987); Pfister, et al. Adv. Cancer Res 48,113-147 (1987)); dsRNA viruses (e.g., reovirus); (+)ssRNA viruses (e.g.,picornavirus, coxsackie virus, hepatitis A virus, poliovirus, togavirus,rubella virus, flavivirus, hepatitis C virus, yellow fever virus, denguevirus, west Nile virus); (−)ssRNA viruses (e.g., orthomyxovirus,influenza virus, rhabdovirus, paramyxovirus, measles virus, mumps virus,parainfluenza virus, respiratory syncytial virus, rhabdovirus, rabiesvirus); ssRNA-RT viruses (e.g. retrovirus, human immunodeficiency virus(HIV)); and, dsDNA-RT viruses (e.g. hepadnavirus, hepatitis B).Immunogens may also be derived from other viruses not listed above butavailable to one of skill in the art.

With respect to HIV, immunogens may be selected from any HIV isolate. Asis well-known in the art, HIV isolates are now classified into discretegenetic subtypes. HIV-1 is known to comprise at least ten subtypes (A1,A2, A3, A4, B, C, D, E, F1, F2, G, H, J and K) (Taylor et al, NEJM,359(18):1965-1966 (2008)). HIV-2 is known to include at least fivesubtypes (A, B, C, D, and E). Subtype B has been associated with the HIVepidemic in homosexual men and intravenous drug users worldwide. MostHIV-1 immunogens, laboratory adapted isolates, reagents and mappedepitopes belong to subtype B. In sub-Saharan Africa, India and China,areas where the incidence of new HIV infections is high, HIV-1 subtype Baccounts for only a small minority of infections, and subtype HIV-1 Cappears to be the most common infecting subtype. Thus, in certainembodiments, it may be preferable to select immunogens from HIV-1subtypes B and/or C. It may be desirable to include immunogens frommultiple HIV subtypes (e.g., HIV-1 subtypes B and C, HIV-2 subtypes Aand B, or a combination of HIV-1 and HIV-2 subtypes) in a singleimmunological composition. Suitable HIV immunogens include ENV, GAG,POL, NEF, as well as variants, derivatives, and fusion proteins thereof,as described by, for example, Gomez et al. Vaccine, Vol. 25, pp.1969-1992 (2007)). Exemplary, suitable peptide immunogens derived fromHIV include but are not limited to VGNLWVTVYYGVPVW (SEQ ID NO. 31),WVTVYYGVPVWKGAT (SEQ ID NO. 32), GATTTLFCASDAKAY (SEQ ID NO. 33),TTLFCASDAKAYDTE (SEQ ID NO. 34), THACVPADPNPQEMV (SEQ ID NO. 35),ENVTENFNMWKNEMV (SEQ ID NO. 36), ENFNMWKNEMVNQMQ (SEQ ID NO. 37),EMVNQMQEDVISLWD (SEQ ID NO. 38), CVKLTPLCVTLECRN (SEQ ID NO. 39),NCSFNATTVVRDRKQ (SEQ ID NO. 40), NATTVVRDRKQTVYA (SEQ ID NO. 41),VYALFYRLDIVPLTK (SEQ ID NO. 42), FYRLDIVPLTKKNYS (SEQ ID NO. 43),INCNTSAITQACPKV (SEQ ID NO. 44), PKVTFDPIPIHYCTP (SEQ ID NO. 45),FDPIPIHYCTPAGYA (SEQ ID NO. 46), TGDIIGDIRQAHCNI (SEQ ID NO. 47),SSSIITIPCRIKQII (SEQ ID NO. 48), ITIPCRIKQIINMWQ (SEQ ID NO. 49),CRIKQIINMWQEVGR (SEQ ID NO. 50), VGRAMYAPPIKGNIT (SEQ ID NO. 51),MYAPPIKGNITCKSN (SEQ ID NO. 52), PIKGNITCKSNITGL (SEQ ID NO. 53),ETFRPGGGDMRNNWR (SEQ ID NO. 54), ELYKYKVVEIKPLGV (SEQ ID NO. 55),YKVVEIKPLGVAPTT (SEQ ID NO. 56), EIKPLGVAPTTTKRR (SEQ ID NO. 57),LGVAPTTTKRRVVER (SEQ ID NO. 58), and/or YSENSSEYY (SEQ ID NO. 59). Anyof these may be encoded by a polynucleotide within a recombinant vector,and/or used in combination with a recombinant vector as part of animmunization strategy.

Immunogens may also be derived from or direct an immune response againstone or more bacterial species (spp.) (e.g., bacterial target antigen(s))including, for example, Bacillus spp. (e.g., Bacillus anthracis),Bordetella spp. (e.g., Bordetella pertussis), Borrelia spp. (e.g.,Borrelia burgdorferi), Brucella spp. (e.g., Brucella abortus, Brucellacanis, Brucella melitensis, Brucella suis), Campylobacter spp. (e.g.,Campylobacter jejuni), Chlamydia spp. (e.g., Chlamydia pneumoniae,Chlamydia psittaci, Chlamydia trachomatis), Clostridium spp. (e.g.,Clostridium botulinum, Clostridium difficile, Clostridium perfringens,Clostridium tetani), Corynebacterium spp. (e.g., Corynebacteriumdiptheriae), Enterococcus spp. (e.g., Enterococcus faecalis,enterococcus faecum), Escherichia spp. (e.g., Escherichia coli),Francisella spp. (e.g., Francisella tularensis), Haemophilus spp. (e.g.,Haemophilus influenza), Helicobacter spp. (e.g., Helicobacter pylori),Legionella spp. (e.g., Legionella pneumophila), Leptospira spp. (e.g.,Leptospira interrogans), Listeria spp. (e.g., Listeria monocytogenes),Mycobacterium spp. (e.g., Mycobacterium leprae, Mycobacteriumtuberculosis), Mycoplasma spp. (e.g., Mycoplasma pneumoniae), Neisseriaspp. (e.g., Neisseria gonorrhea, Neisseria meningitidis), Pseudomonasspp. (e.g., Pseudomonas aeruginosa), Rickettsia spp. (e.g., Rickettsiarickettsii), Salmonella spp. (e.g., Salmonella typhi, Salmonellatyphinurium), Shigella spp. (e.g., Shigella sonnei), Staphylococcus spp.(e.g., Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcussaprophyticus, coagulase negative staphylococcus (e.g., U.S. Pat. No.7,473,762)), Streptococcus spp. (e.g., Streptococcus agalactiae,Streptococcus pneumoniae, Streptococcus pyrogenes), Treponema spp.(e.g., Treponema pallidum), Vibrio spp. (e.g., Vibrio cholerae), andYersinia spp. (Yersinia pestis). Immunogens may also be derived from ordirect the immune response against other bacterial species not listedabove but available to one of skill in the art.

Immunogens may also be derived from or direct an immune response againstone or more parasitic organisms (spp.) (e.g., parasite targetantigen(s)) including, for example, Ancylostoma spp. (e.g., A.duodenale), Anisakis spp., Ascaris lumbricoides, Balantidium coli,Cestoda spp., Cimicidae spp., Clonorchis sinensis, Dicrocoeliumdendriticum, Dicrocoelium hospes, Diphyllobothrium latum, Dracunculusspp., Echinococcus spp. (e.g., E. granulosus, E. multilocularis),Entamoeba histolytica, Enterobius vermicularis, Fasciola spp. (e.g., F.hepatica, F. magna, F. gigantica, F. jacksoni), Fasciolopsis buski,Giardia spp. (Giardia lamblia), Gnathostoma spp., Hymenolepis spp.(e.g., H. nana, H. diminuta), Leishmania spp., Loa loa, Metorchis spp.(M. conjunctus, M. albidus), Necator americanus, Oestroidea spp. (e.g.,botfly), Onchocercidae spp., Opisthorchis spp. (e.g., O. viverrini, O.felineus, O. guayaquilensis, and O. noverca), Plasmodium spp. (e.g., P.falciparum), Protofasciola robusta, Parafasciolopsis fasciomorphae,Paragonimus westermani, Schistosoma spp. (e.g., S. mansoni, S.japonicum, S. mekongi, S. haematobium), Spirometra erinaceieuropaei,Strongyloides stercoralis, Taenia spp. (e.g., T. saginata, T. solium),Toxocara spp. (e.g., T. canis, T. cati), Toxoplasma spp. (e.g., T.gondii), Trichobilharzia regenti, Trichinella spiralis, Trichuristrichiura, Trombiculidae spp., Trypanosoma spp., Tunga penetrans, and/orWuchereria bancrofti. Immunogens may also be derived from or direct theimmune response against other parasitic organisms not listed above butavailable to one of skill in the art.

Immunogens may be derived from or direct the immune response againsttumor target antigens (e.g., tumor target antigens). The term tumortarget antigen (TA) may include both tumor-associated antigens (TAAs)and tumor-specific antigens (TSAs), where a cancerous cell is the sourceof the antigen. A TA may be an antigen that is expressed on the surfaceof a tumor cell in higher amounts than is observed on normal cells or anantigen that is expressed on normal cells during fetal development. ATSA is typically an antigen that is unique to tumor cells and is notexpressed on normal cells. TAs are typically classified into fivecategories according to their expression pattern, function, or geneticorigin: cancer-testis (CT) antigens (i.e., MAGE, NY-ESO-1); melanocytedifferentiation antigens (i.e., Melan A/MART-1, tyrosinase, gp100);mutational antigens (i.e., MUM-1, p53, CDK-4); overexpressed ‘self’antigens (i.e., HER-2/neu, p53); and, viral antigens (i.e., HPV, EBV).Suitable TAs include, for example, gp100 (Cox et al., Science,264:716-719 (1994)), MART-1/Melan A (Kawakami et al., J. Exp. Med.,180:347-352 (1994)), gp75 (TRP-1) (Wang et al., J. Exp. Med.,186:1131-1140 (1996)), tyrosinase (Wolfel et al., Eur. J. Immunol.,24:759-764 (1994)), NY-ESO-1 (WO 98/14464; WO 99/18206), melanomaproteoglycan (Hellstrom et al., J. Immunol., 130:1467-1472 (1983)), MAGEfamily antigens (i.e., MAGE-1, 2,3,4,6, and 12; Van der Bruggen et al.,Science, 254:1643-1647 (1991); U.S. Pat. Nos. 6,235,525), BAGE familyantigens (Boel et al., Immunity, 2:167-175 (1995)), GAGE family antigens(i.e., GAGE-1,2; Van den Eynde et al., J. Exp. Med., 182:689-698 (1995);U.S. Pat. No. 6,013,765), RAGE family antigens (i.e., RAGE-1; Gaugler etat., Immunogenetics, 44:323-330 (1996); U.S. Pat. No. 5,939,526),N-acetylglucosaminyltransferase-V (Guilloux et at., J. Exp. Med.,183:1173-1183 (1996)), p15 (Robbins et al., J. Immunol. 154:5944-5950(1995)), β-catenin (Robbins et al., J. Exp. Med., 183:1185-1192 (1996)),MUM-1 (Coulie et al., Proc. Natl. Acad. Sci. USA, 92:7976-7980 (1995)),cyclin dependent kinase-4 (CDK4) (Wolfel et al., Science, 269:1281-1284(1995)), p21-ras (Fossum et at., Int. J. Cancer, 56:40-45 (1994)),BCR-abl (Bocchia et al., Blood, 85:2680-2684 (1995)), p53 (Theobald etal., Proc. Natl. Acad. Sci. USA, 92:11993-11997 (1995)), p185 HER2/neu(erb-B1; Fisk et al., J. Exp. Med., 181:2109-2117 (1995)), epidermalgrowth factor receptor (EGFR) (Harris et al., Breast Cancer Res. Treat,29:1-2 (1994)), carcinoembryonic antigens (CEA) (Kwong et al., J. Natl.Cancer Inst., 85:982-990 (1995) U.S. Pat. Nos. 5,756,103; 5,274,087;5,571,710; 6,071,716; 5,698,530; 6,045,802; EP 263933; EP 346710; and,EP 784483); carcinoma-associated mutated mucins (i.e., MUC-1 geneproducts; Jerome et al., J. Immunol., 151:1654-1662 (1993)); EBNA geneproducts of EBV (i.e., EBNA-1; Rickinson et al., Cancer Surveys,13:53-80 (1992)); E7, E6 proteins of human papillomavirus (Ressing etal., J. Immunol, 154:5934-5943 (1995)); prostate specific antigen (PSA;Xue et al., The Prostate, 30:73-78 (1997)); prostate specific membraneantigen (PSMA; Israeli, et al., Cancer Res., 54:1807-1811 (1994));idiotypic epitopes or antigens, for example, immunoglobulin idiotypes orT cell receptor idiotypes (Chen et al., J. Immunol., 153:4775-4787(1994)); KSA (U.S. Pat. No. 5,348,887), kinesin 2 (Dietz, et al. BiochemBiophys Res Commun 2000 Sep. 7; 275(3):731-8), HIP-55, TGFβ-1anti-apoptotic factor (Toomey, et al. Br J Biomed Sci 2001;58(3):177-83), tumor protein D52 (Bryne J. A., et al., Genomics,35:523-532 (1996)), H1FT, NY-BR-1 (WO 01/47959), NY-BR-62, NY-BR-75,NY-BR-85, NY-BR-87 and NY-BR-96 (Scanlan, M. Serologic and BioinformaticApproaches to the Identification of Human Tumor Antigens, in CancerVaccines 2000, Cancer Research Institute, New York, N.Y.), and/orpancreatic cancer antigens (e.g., SEQ ID NOS: 1-288 of U.S. Pat. No.7,473,531). Immunogens may also be derived from or direct the immuneresponse against include TAs not listed above but available to one ofskill in the art.

In some embodiments, derivatives of polypeptides, peptides, orpolynucleotides incorporated into or expressed by the vectors describedherein including, for example, fragments and/or variants thereof may beutilized. Derivatives may result from, for example, substitution,deletion, or addition of amino acids or nucleotides from or to thereference sequence (e.g., the parental sequence). A derivative of apolypeptide or protein, for example, typically refers to an amino acidsequence that is altered with respect to the referenced polypeptide orpeptide. A derivative of a polypeptide typically retains at least oneactivity of the polypeptide. A derivative will typically share at leastapproximately 60%, 70%, 80%, 90%, 95%, or 99% identity to the referencesequence. With respect to polypeptides and peptides, the derivative mayhave “conservative” changes, wherein a substituted amino acid hassimilar structural or chemical properties. A derivative may also have“nonconservative” changes. Exemplary, suitable conservative amino acidsubstitutions may include, for example, those shown in Table 1:

TABLE 1 Original Preferred Residues Exemplary SubstitutionsSubstitutions Ala Val, Leu, Ile Val Arg Lys, Gln, Asn Lys Asn Gln GlnAsp Glu Glu Cys Ser, Ala Ser Gln Asn Asn Glu Asp Asp Gly Pro, Ala AlaHis Asn, Gln, Lys, Arg Arg Ile Leu, Val, Met, Ala, Phe, Norleucine LeuLeu Norleucine, Ile, Val, Met, Ala, Phe Ile Lys Arg, 1,4 Diamino-butyricAcid, Gln, Asn Arg Met Leu, Phe, Ile Leu Phe Leu, Val, Ile, Ala, Tyr LeuPro Ala Gly Ser Thr, Ala, Cys Thr Thr Ser Ser Trp Tyr, Phe Tyr Tyr Trp,Phe, Thr, Ser Phe Val Ile, Met, Leu, Phe, Ala, Norleucine LeuOther amino acid substitutions may be considered non-conservative.Derivatives may also include amino acid or nucleotide deletions and/oradditions/insertions, or some combination of these. Guidance indetermining which amino acid residues or nucleotides may be substituted,inserted, or deleted without abolishing the desired activity of thederivative may be identified using any of the methods available to oneof skill in the art.

Derivatives may also refer to a chemically modified polynucleotide orpolypeptide. Chemical modifications of a polynucleotide may include, forexample, replacement of hydrogen by an alkyl, acyl, hydroxyl, or aminogroup. A derivative polynucleotide may encode a polypeptide whichretains at least one biological or immunological function of the naturalmolecule. A derivative polypeptide may be one modified by glycosylation,pegylation, biotinylation, or any similar process that retains at leastone biological or immunological function of the polypeptide from whichit was derived.

The phrases “percent identity” and “% identity,” as applied topolypeptide sequences, refer to the percentage of residue matchesbetween at least two polypeptide sequences aligned using a standardizedalgorithm. Methods of polypeptide sequence alignment are well-known.Some alignment methods take into account conservative amino acidsubstitutions. Such conservative substitutions, explained in more detailabove, generally preserve the charge and hydrophobicity at the site ofsubstitution, thus preserving the structure (and therefore function) ofthe polypeptide. Percent identity may be measured over the length of anentire defined polypeptide sequence, for example, as defined by aparticular SEQ ID number, or may be measured over a shorter length, forexample, over the length of a fragment taken from a larger, definedpolypeptide sequence, for instance, a fragment of at least 10, at least15, at least 20, at least 30, at least 40, at least 50, at least 70 orat least 150 contiguous residues. Such lengths are exemplary only, andit is understood that any fragment length supported by the sequencesshown herein, in the tables, figures or Sequence Listing, may be used todescribe a length over which percentage identity may be measured.

As mentioned above, this disclosure relates to compositions comprisingrecombinant vectors, the vectors per se, and methods of using the same.A “vector” is any moiety (e.g., a virus or plasmid) used to carry,introduce, or transfer a polynucleotide or interest to another moiety(e.g., a host cell). In certain cases, an expression vector is utilized.An expression vector is a nucleic acid molecule containing apolynucleotide of interest encoding a polypeptide, peptide, orpolynucleotide and also containing other polynucleotides that directand/or control the expression of the polynucleotide of interest.Expression includes, but is not limited to, processes such astranscription, translation, and/or splicing (e.g., where introns arepresent).

Viral vectors that may be used include, for example, retrovirus,adenovirus, adeno-associated virus (AAV), alphavirus, herpes virus, andpoxvirus vectors, among others. Many such viral vectors are available inthe art. The vectors described herein may be constructed using standardrecombinant techniques widely available to one skilled in the art. Suchtechniques may be found in common molecular biology references such asMolecular Cloning: A Laboratory Manual (Sambrook, et al., 1989, ColdSpring Harbor Laboratory Press), Gene Expression Technology (Methods inEnzymology, Vol. 185, edited by D. Goeddel, 1991. Academic Press, SanDiego, Calif.), and PCR Protocols: A Guide to Methods and Applications(Innis, et al. 1990. Academic Press, San Diego, Calif.).

Suitable retroviral vectors may include derivatives of lentivirus aswell as derivatives of murine or avian retroviruses. Examplary, suitableretroviral vectors may include, for example, Moloney murine leukemiavirus (MoMuLV), Harvey murine sarcoma virus (HaMuSV), murine mammarytumor virus (MuMTV), SIV, BIV, HIV and Rous Sarcoma Virus (RSV). Anumber of retroviral vectors can incorporate multiple exogenouspolynucleotides. As recombinant retroviruses are defective, they requireassistance in order to produce infectious vector particles. Thisassistance can be provided by, for example, helper cell lines encodingretrovirus structural genes. Suitable helper cell lines include Ψ2,PA317 and PA12, among others. The vector virions produced using suchcell lines may then be used to infect a tissue cell line, such as NIH3T3 cells, to produce large quantities of chimeric retroviral virions.Retroviral vectors may be administered by traditional methods (i.e.,injection) or by implantation of a “producer cell line” in proximity tothe target cell population (Culver, K., et al., 1994, Hum. Gene Ther., 5(3): 343-79; Culver, K., et al., Cold Spring Harb. Symp. Quant. Biol.,59: 685-90); Oldfield, E., 1993, Hum. Gene Ther., 4 (1): 39-69). Theproducer cell line is engineered to produce a viral vector and releasesviral particles in the vicinity of the target cell. A portion of thereleased viral particles contact the target cells and infect thosecells, thus delivering a nucleic acid encoding an immunogen to thetarget cell. Following infection of the target cell, expression of thepolynucleotide of interest from the vector occurs.

Adenoviral vectors have proven especially useful for gene transfer intoeukaryotic cells (Rosenfeld, M., et al., 1991, Science, 252 (5004):431-4; Crystal, R., et al., 1994, Nat. Genet., 8 (1): 42-51), the studyeukaryotic gene expression (Levrero, M., et al., 1991, Gene, 101 (2):195-202), vaccine development (Graham, F. and Prevec, L., 1992,Biotechnology, 20: 363-90), and in animal models (Stratford-Perricaudet,L., et al., 1992, Bone Marrow Transplant., 9 (Suppl. 1): 151-2; Rich, etal., 1993, Hum. Gene Ther., 4 (4): 461-76). Experimental routes foradministrating recombinant Ad to different tissues in vivo have includedintratracheal instillation (Rosenfeld, M., et al., 1992, Cell, 68 (1):143-55) injection into muscle (Quantin, B., et al., 1992, Proc. Natl.Acad. Sci. U.S.A., 89 (7): 2581-4), peripheral intravenous injection(Herz, J., and Gerard, R., 1993, Proc. Natl. Acad. Sci. U.S.A., 90 (7):2812-6) and/or stereotactic inoculation to brain (Le Gal La SalIe, G.,et al., 1993, Science, 259 (5097): 988-90), among others.

Adeno-associated virus (AAV) demonstrates high-level infectivity, broadhost range and specificity in integrating into the host cell genome(Hermonat, P., et al., 1984, Proc. Natl. Acad. Sci. U.S.A., 81 (20):6466-70). And Herpes Simplex Virus type-1 (HSV-1) is yet anotherattractive vector system, especially for use in the nervous systembecause of its neurotropic property (Geller, A., et al., 1991, TrendsNeurosci., 14 (10): 428-32; Glorioso, et al., 1995, Mol. Biotechnol., 4(1): 87-99; Glorioso, et al., 1995, Annu. Rev. Microbiol., 49: 675-710).

Alphavirus may also be used to express the immunogen in a host. Suitablemembers of the Alphavirus genus include, among others, Sindbis virus,Semliki Forest virus (SFV), the Ross River virus and Venezuelan, Westernand Eastern equine encephalitis viruses, among others. Expressionsystems utilizing alphavirus vectors are described in, for example, U.S.Pat. Nos. 5,091,309; 5,217,879; 5,739,026; 5,766,602; 5,843,723;6,015,694; 6,156,558; 6,190,666; 6,242,259; and, 6,329,201; WO 92/10578;Xiong et al., Science, Vol 243, 1989, 1188-1191; Liliestrom, et al.Bio/Technology, 9: 1356-1361, 1991. Thus, the use of alphavirus as anexpression system is well known by those of skill in the art.

Poxvirus is another useful expression vector (Smith, et al. 1983, Gene,25 (1): 21-8; Moss, et al, 1992, Biotechnology, 20: 345-62; Moss, et al,1992, Curr. Top. Microbiol. Immunol., 158: 25-38; Moss, et al. 1991.Science, 252: 1662-1667). The most often utilized poxviral vectorsinclude vaccinia and derivatives therefrom such as NYVAC and MVA, andmembers of the avipox genera such as fowlpox, canarypox, ALVAC, andALVAC(2), among others.

An exemplary suitable vector is NYVAC (vP866) which was derived from theCopenhagen vaccine strain of vaccinia virus by deleting six nonessentialregions of the genome encoding known or potential virulence factors(see, for example, U.S. Pat. Nos. 5,364,773 and 5,494,807). The deletionloci were also engineered as recipient loci for the insertion of foreigngenes. The deleted regions are: thymidine kinase gene (TK; J2R);hemorrhagic region (u; B13R+B14R); A type inclusion body region (ATI;A26L); hemagglutinin gene (HA; A56R); host range gene region (C7L-K1L);and, large subunit, ribonucleotide reductase (I4L). NYVAC is agenetically engineered vaccinia virus strain that was generated by thespecific deletion of eighteen open reading frames encoding gene productsassociated with virulence and host range. NYVAC has been show to beuseful for expressing TAs (see, for example, U.S. Pat. No. 6,265,189).NYVAC (vP866), vP994, vCP205, vCP1433, placZH6H4Lreverse, pMPC6H6K3E3and pC3H6FHVB were also deposited with the ATCC under the terms of theBudapest Treaty, accession numbers VR-2559, VR-2558, VR-2557, VR-2556,ATCC-97913, ATCC-97912, and ATCC-97914, respectively.

Another suitable virus is the Modified Vaccinia Ankara (MVA) virus whichwas generated by 516 serial passages on chicken embryo fibroblasts ofthe Ankara strain of vaccinia virus (CVA) (for review see Mayr, A., etal. Infection 3, 6-14 (1975)). It was shown in a variety of animalmodels that the resulting MVA was significantly avirulent (Mayr, A. &Danner, K. [1978] Dev. Biol. Stand. 41: 225.34) and has been tested inclinical trials as a smallpox vaccine (Mayr et al., Zbl. Bakt. Hyg. I,Abt. Org. B 167, 375-390 (1987), Stickl et al., Dtsch. med. Wschr. 99,2386-2392 (1974)). MVA has also been engineered for use as a viralvector for both recombinant gene expression studies and as a recombinantvaccine (Sutter, G. et al. (1994), Vaccine 12: 1032-40; Blanchard etal., 1998, J Gen Virol 79, 1159-1167; Carroll & Moss, 1997, Virology238, 198-211; Altenberger, U.S. Pat. No. 5,185,146; Ambrosini et al.,1999, J Neurosci Res 55(5), 569). Modified virus Ankara (MVA) has beenpreviously described in, for example, U.S. Pat. Nos. 5,185,146 and6,440,422; Sutter, et al. (B. Dev. Biol. Stand. Basel, Karger 84:195-200(1995)); Antoine, et al. (Virology 244: 365-396, 1998); Sutter et al.(Proc. Natl. Acad. Sci. USA 89: 10847-10851, 1992); Meyer et al. (J.Gen. Virol. 72: 1031-1038, 1991); Mahnel, ett al. (Berlin Munch.Tierarztl. Wochenschr. 107: 253-256, 1994); Mayr et al. (Zbl. Bakt. Hyg.I, Abt. Org. B 167: 375-390 (1987); and, Stickl et al. (Dtsch. med.Wschr. 99: 2386-2392 (1974)). An exemplary MVA is available from theATCC under accession numbers VR-1508 and VR-1566.

ALVAC-based recombinant viruses (i.e., ALVAC-1 and ALVAC-2) are alsosuitable for use in practicing the present invention (see, for example,U.S. Pat. No. 5,756,103). ALVAC(2) is identical to ALVAC(1) except thatALVAC(2) genome comprises the vaccinia E3L and K3L genes under thecontrol of vaccinia promoters (U.S. Pat. No. 6,130,066; Beattie et al.,1995a, 1995b, 1991; Chang et al., 1992; Davies et al., 1993). BothALVAC(1) and ALVAC(2) have been demonstrated to be useful in expressingforeign DNA sequences, such as TAs (Tartaglia et al., 1993 a,b; U.S.Pat. No. 5,833,975). ALVAC was deposited under the terms of the BudapestTreaty with the American Type Culture Collection (ATCC), 10801University Boulevard, Manassas, Va. 20110-2209, USA, ATCC accessionnumber VR-2547. Vaccinia virus host range genes (e.g., C18L, C17L, C7L,K1L, E3L, B4R, B23R, and B24R) have also been shown to be expressible incanarypox (e.g., U.S. Pat. No. 7,473,536).

Another useful poxvirus vector is TROVAC. TROVAC refers to an attenuatedfowlpox that was a plaque-cloned isolate derived from the FP-1 vaccinestrain of fowlpoxvirus which is licensed for vaccination of 1 day oldchicks. TROVAC was likewise deposited under the terms of the BudapestTreaty with the ATCC, accession number 2553.

“Non-viral” plasmid vectors may also be suitable for use. Plasmid DNAmolecules comprising expression cassettes for expressing an immunogenmay be used for “naked DNA” immunization. Preferred plasmid vectors arecompatible with bacterial, insect, and/or mammalian host cells. Suchvectors include, for example, PCR-II, pCR3, and pcDNA3.1 (Invitrogen,San Diego, Calif.), pBSII (Stratagene, La Jolla, Calif.), pET15(Novagen, Madison, Wis.), pGEX (Pharmacia Biotech, Piscataway, N.J.),pEGFP-N2 (Clontech, Palo Alto, Calif.), pETL (BlueBacII, Invitrogen),pDSR-alpha (PCT pub. No. WO 90/14363) and pFastBacDual (Gibco-BRL, GrandIsland, N.Y.) as well as Bluescript® plasmid derivatives (a high copynumber COLE1-based phagemid, Stratagene Cloning Systems, La Jolla,Calif.), PCR cloning plasmids designed for cloning Taq-amplified PCRproducts (e.g., TOPO™ TA Cloning® kit, PCR2.1° plasmid derivatives,Invitrogen, Carlsbad, Calif.).

Bacterial vectors may also be suitable for use. These vectors include,for example, Shigella, Salmonella, Vibrio cholerae, Lactobacillus,Bacille calmette guérin (BCG), and Streptococcus (see for example, WO88/6626; WO 90/0594; WO 91/13157; WO 92/1796; and WO 92/21376). Manyother non-viral plasmid expression vectors and systems are known in theart and could be used with the current invention.

The polynucleotides and polypeptides referred to herein as beingsuitable for use and/or modification (e.g., SEQ ID NOS. 1-28) may beinserted into non-homologous vector genomes. For instance, while thepolynucleotides and polypeptides of SEQ ID NOS. 1-28 may be derived fromvaccinia, any one or more of such polynucleotides and/or polypeptidesmay be incorporated into and/or expressed within a different viral(e.g., MVA, ALVAC, ALVAC(2), TROVAC), bacterial or plasmid vector. Ifsuch different vectors contain sequence homologous to one or more of SEQID NOS. 1-28, such sequence may be replaced by a polynucleotide encodingSEQ ID NOS. 1-28. Such vectors may further comprise or be modified tocomprise a polynucleotide encoding SEQ ID NO. 29, such as SEQ ID NO. 30.

Expression vectors typically comprise one or more flankingpolynucleotides “operably linked” to a heterologous polynucleotideencoding a polypeptide. As used herein, the term “operably linked”refers to a linkage between polynucleotide elements in a functionalrelationship such as when promoter or enhancer affects transcription ofa polynucleotide of interest (e.g., a coding sequence). Flankingpolynucleotides may be homologous (e.g., from the same species and/orstrain as the host cell), heterologous (e.g., from a species other thanthe host cell species and/or strain), hybrid (e.g., a combination offlanking sequences from more than one source), or synthetic, forexample. All polynucleotides referred to herein are typicallyincorporated into vectors in expressible form, meaning that suchpolynucleotides are capable of being expressed from the expressionvector transformed into a cell or after incorporation of the expressionvector or portions thereof into the genome of an infected or transformedcell, such that the polypeptide encoded thereby is expressed in theinfected or transformed cell. The flanking sequences described hereintypically assist in achieving expression in the infected or transformedcell.

In certain embodiments, it is preferred that the flanking polynucleotideincludes a transcriptional regulatory region that drives expression of apolynucleotide of interest in an environment such as a target cell. Thetranscriptional regulatory region may comprise, for example, a promoter,enhancer, silencer, repressor element, or combinations thereof. Thetranscriptional regulatory region may be either constitutive,tissue-specific, cell-type specific (e.g., the region is drives higherlevels of transcription in a one type of tissue or cell as compared toanother) and/or regulatable (e.g., responsive to interaction with acompound such as tetracycline). The source of a transcriptionalregulatory region may be any prokaryotic or eukaryotic organism, anyvertebrate or invertebrate organism, or any plant, provided that theflanking polynucleotide functions in an environment (e.g., a cell) bycausing transcription of a polynucleotide within that environment. Awide variety of suitable transcriptional regulatory regions areavailable to one of skill in the art.

Suitable transcriptional regulatory regions include, for example, thesynthetic e/1 promoter; the CMV promoter (e.g., the CMV-immediate earlypromoter); promoters from eukaryotic genes (e.g., the estrogen-induciblechicken ovalbumin gene, the interferon genes, thegluco-corticoid-inducible tyrosine aminotransferase gene, and thethymidine kinase gene); and the major early and late adenovirus genepromoters; the sv40 early promoter region (Bernoist, et al. Nature290:304-10 (1981)); the promoter contained in the 3′ long terminalrepeat (LTR) of Rous sarcoma virus (RSV) (Yamamoto, et al., 1980, cell22:787-97); the herpes simplex virus thymidine kinase (HSV-TK) promoter(Wagner et al., Proc. Natl. Acad. Sci. USA, 78:1444-45 (1981)); theregulatory sequences of the metallothionine gene (Brinster et al. Nature296:39-42 (1982)); prokaryotic expression vectors such as thebeta-lactamase promoter (Villa-kamaroff et al., Proc. Natl. Acad. Sci.USA, 75:3727-31 (1978)); or, the tac promoter (Deboer et al. Proc. Natl.Acad. Sci. U.s.a., 80:21-25 (1983)). Tissue- and/or cell-type specifictranscriptional control regions include, for example, the elastase Igene control region which is active in pancreatic acinar cells (Swift etal. Cell 38:639-46 (1984); Ornitz, et al. Cold Spring Harbor Symp.Quant. Biol. 50:399-409 (1986); Macdonald, et al. Hepatology 7:425-515(1987)); the insulin gene control region which is active in pancreaticbeta cells (Hanahan, et al. Nature 315:115-22 (1985)); theimmunoglobulin gene control region which is active in lymphoid cells(Grosschedl et al. Cell 38:647-58 (1984); Adames et al. Nature318:533-38 (1985); Alexander et al., Mol. Cell. Biol., 7:1436-44(1987)); the mouse mammary tumor virus control region in testicular,breast, lymphoid and mast cells (Leder et al. Cell 45:485-95 (1986));the albumin gene control region in liver (Pinkert et al. Genes andDevel. 1:268-76 (1987)); the alpha-feto-protein gene control region inliver (Krumlauf et al. Mol. Cell. Biol., 5:1639-48 (1985); Hammer et al.Science 235:53-58 (1987)); the alpha 1-antitrypsin gene control regionin liver (Kelsey et al. Genes and Devel. 1:161-71 (1987)); thebeta-globin gene control region in myeloid cells (Mogram et al. Nature315:338-40 (1985); Kollias et al. Cell 46:89-94 (1986)); the myelinbasic protein gene control region in oligodendrocyte cells in the brain(Readhead et al. Cell 48:703-12 (1987)); the myosin light chain-2 genecontrol region in skeletal muscle (Sani, et al. Nature 314:283-86(1985)); the gonadotropic releasing hormone gene control region in thehypothalamus (Mason et al. Science 234:1372-78 (1986)), and thetyrosinase promoter in melanoma cells (Hart, et al. Semin. Oncol.February; 23(1):154-8 (1996); Siders, et al. Cancer Gene Ther.September-October, 5(5):281-91 (1998)), among others. Other suitablepromoters are known in the art.

Nucleic acid delivery or transformation techniques that may be usedinclude DNA-ligand complexes, adenovirus-ligand-DNA complexes, directinjection of DNA, CaPO₄ precipitation, gene gun techniques,electroporation, and colloidal dispersion systems, among others.Colloidal dispersion systems include macromolecule complexes,nanocapsules, microspheres, beads, and lipid-based systems includingoil-in-water emulsions, micelles, mixed micelles, and liposomes. Thepreferred colloidal system of this invention is a liposome, which areartificial membrane vesicles useful as delivery vehicles in vitro and invivo. RNA, DNA and intact virions can be encapsulated within the aqueousinterior and be delivered to cells in a biologically active form(Fraley, R., et al. Trends Biochem. Sci., 6: 77 (1981)). The compositionof the liposome is usually a combination of phospholipids, particularlyhigh-phase-transition-temperature phospholipids, usually in combinationwith steroids, especially cholesterol. Other phospholipids or otherlipids may also be used. The physical characteristics of liposomesdepend on pH, ionic strength, and the presence of divalent cations.Examples of lipids useful in liposome production include phosphatidylcompounds, such as phosphatidylglycerol, phosphatidylcholine,phosphatidylserine, phosphatidyletha-nolamine, sphingolipids,cerebrosides, and gangliosides. Particularly useful arediacylphosphatidylglycerols, where the lipid moiety contains from 14-18carbon atoms, particularly from 16-18 carbon atoms, and is saturated.Illustrative phospholipids include egg phosphatidylcholine,dipalmitoylphosphatidylcholine and di stearoylphosphatidylcholine.

Strategies for improving the efficiency of nucleic acid-basedimmunization may also be used including, for example, the use ofself-replicating viral replicons (Caley, et al. Vaccine, 17: 3124-2135(1999); Dubensky, et al. Mol. Med. 6: 723-732 (2000); Leitner, et al.Cancer Res. 60: 51-55 (2000)), codon optimization (Liu, et al. Mol.Ther., 1: 497-500 (2000); Dubensky, supra; Huang, et al. J. Virol. 75:4947-4951 (2001)), in vivo electroporation (Widera, et al. J. Immunol.164: 4635-3640 (2000)), incorporation of CpG stimulatory motifs(Gurunathan, et al. Ann. Rev. Immunol. 18: 927-974 (2000); Leitner,supra), sequences for targeting of the endocytic or ubiquitin-processingpathways (Thomson, et al. J. Virol. 72: 2246-2252 (1998); Velders, etal. J. Immunol. 166: 5366-5373 (2001)), prime-boost regimens(Gurunathan, supra; Sullivan, et al. Nature, 408: 605-609 (2000); Hanke,et al. Vaccine, 16: 439-445 (1998); Amara, et al. Science, 292: 69-74(2001)), and the use of mucosal delivery vectors such as Salmonella(Darji, et al. Cell, 91: 765-775 (1997); Woo, et al. Vaccine, 19:2945-2954 (2001)). Other methods are known in the art, some of which aredescribed below.

In other embodiments, it may be advantageous to combine or includewithin the compositions or recombinant vectors additional polypeptides,peptides or polynucleotides encoding one or more polypeptides orpeptides that function as “co-stimulatory” component(s). Suchco-stimulatory components may include, for example, cell surfaceproteins, cytokines or chemokines in a composition of the presentinvention. The co-stimulatory component may be included in thecomposition as a polypeptide or peptide, or as a polynucleotide encodingthe polypeptide or peptide, for example. Suitable co-stimulatorymolecules may include, for example, polypeptides that bind members ofthe CD28 family (i.e., CD28, ICOS; Hutloff, et al. Nature 1999, 397:263-265; Peach, et al. J Exp Med 1994, 180: 2049-2058) such as the CD28binding polypeptides B7.1 (CD80; Schwartz, 1992; Chen et al, 1992;Ellis, et al. J. Immunol., 156(8): 2700-9) and B7.2 (CD86; Ellis, et al.J. Immunol., 156(8): 2700-9); polypeptides which bind members of theintegrin family (i.e., LFA-1 (CD11a/CD18); Sedwick, et al. J Immunol1999, 162: 1367-1375; Wülfing, et al. Science 1998, 282: 2266-2269; Lub,et al. Immunol Today 1995, 16: 479-483) including members of the ICAMfamily (i.e., ICAM-1, -2 or -3); polypeptides which bind CD2 familymembers (i.e., CD2, signalling lymphocyte activation molecule (CDw150 or“SLAM”; Aversa, et al. J Immunol 1997, 158: 4036-4044) such as CD58(LFA-3; CD2 ligand; Davis, et al. Immunol Today 1996, 17: 177-187) orSLAM ligands (Sayos, et al. Nature 1998, 395: 462-469); polypeptideswhich bind heat stable antigen (HSA or CD24; Zhou, et al. Eur J Immunol1997, 27: 2524-2528); polypeptides which bind to members of the TNFreceptor (TNFR) family (i.e., 4-1BB (CD137; Vinay, et al. Semin Immunol1998, 10: 481-489)), OX40 (CD134; Weinberg, et al. Semin Immunol 1998,10: 471-480; Higgins, et al. J Immunol 1999, 162: 486-493), and CD27(Lens, et al. Semin Immunol 1998, 10: 491-499)) such as 4-1BBL (4-1BBligand; Vinay, et al. Semin Immunol 1998, 10: 481-48; DeBenedette, etal. J Immunol 1997, 158: 551-559), TNFR associated factor-1 (TRAF-1;4-1BB ligand; Saoulli, et al. J Exp Med 1998, 187: 1849-1862, Arch, etal. Mol Cell Biol 1998, 18: 558-565), TRAF-2 (4-1BB and OX40 ligand;Saoulli, et al. J Exp Med 1998, 187: 1849-1862; Oshima, et al. IntImmunol 1998, 10: 517-526, Kawamata, et al. J Biol Chem 1998, 273:5808-5814), TRAF-3 (4-1BB and OX40 ligand; Arch, et al. Mol Cell Biol1998, 18: 558-565; Jang, et al. Biochem Biophys Res Commun 1998, 242:613-620; Kawamata S, et al. J Biol Chem 1998, 273: 5808-5814), OX40L(OX40 ligand; Gramaglia, et al. J Immunol 1998, 161: 6510-6517), TRAF-5(OX40 ligand; Arch, et al. Mol Cell Biol 1998, 18: 558-565; Kawamata, etal. J Biol Chem 1998, 273: 5808-5814), and CD70 (CD27 ligand; Couderc,et al. Cancer Gene Ther., 5(3): 163-75). CD154 (CD40 ligand or “CD40L”;Gurunathan, et al. J. Immunol., 1998, 161: 4563-4571; Sine, et al. Hum.Gene Ther., 2001, 12: 1091-1102) Other co-stimulatory molecules may alsobe suitable for practicing the present invention.

One or more cytokines may also be suitable co-stimulatory components or“adjuvants”, either as polypeptides or being encoded by polynucleotidescontained within the compositions of the present invention (Parmiani, etal. Immunol Lett 2000 Sep. 15; 74(1): 41-4; Berzofsky, et al. NatureImmunol. 1: 209-219). Suitable cytokines include, for example,interleukin-2 (IL-2) (Rosenberg, et al. Nature Med. 4: 321-327 (1998)),IL-4, IL-7, IL-12 (reviewed by Pardoll, 1992; Harries, et al. J. GeneMed. 2000 July-August; 2(4):243-9; Rao, et al. J. Immunol. 156:3357-3365 (1996)), IL-15 (Xin, et al. Vaccine, 17:858-866, 1999), IL-16(Cruikshank, et al. J. Leuk Biol. 67(6): 757-66, 2000), IL-18 (J. CancerRes. Clin. Oncol. 2001. 127(12): 718-726), GM-CSF (CSF (Disis, et al.Blood, 88: 202-210 (1996)), tumor necrosis factor-alpha (TNF-α), orinterferon-gamma (INF-γ). Other cytokines may also be suitable forpracticing the present invention.

Chemokines may also be utilized. For example, fusion proteins comprisingCXCL10 (IP-10) and CCL7 (MCP-3) fused to a tumor self-antigen have beenshown to induce anti-tumor immunity (Biragyn, et al. Nature Biotech.1999, 17: 253-258). The chemokines CCL3 (MIP-1α) and CCLS (RANTES)(Boyer, et al. Vaccine, 1999, 17 (Supp. 2): S53-S64) may also be of use.Other suitable chemokines are known in the art.

It is also known in the art that suppressive or negative regulatoryimmune mechanisms may be blocked, resulting in enhanced immuneresponses. For instance, treatment with anti-CTLA-4 (Shrikant, et al.Immunity, 1996, 14: 145-155; Sutmuller, et al. J. Exp. Med., 2001, 194:823-832), anti-CD25 (Sutmuller, supra), anti-CD4 (Matsui, et al. J.Immunol., 1999, 163: 184-193), the fusion protein IL13Ra2-Fc (Terabe, etal. Nature Immunol., 2000, 1: 515-520), and combinations thereof (i.e.,anti-CTLA-4 and anti-CD25, Sutmuller, supra) have been shown toupregulate anti-tumor immune responses and would be suitable inpracticing the present invention.

An immunogen may also be administered in combination with one or moreadjuvants to boost the immune response. Adjuvants may also be includedto stimulate or enhance the immune response. Non-limiting examples ofsuitable adjuvants include those of the gel-type (i.e., aluminumhydroxide/phosphate (“alum adjuvants”), calcium phosphate), of microbialorigin (muramyl dipeptide (MDP)), bacterial exotoxins (cholera toxin(CT), native cholera toxin subunit B (CTB), E. coli labile toxin (LT),pertussis toxin (PT), CpG oligonucleotides, BCG sequences, tetanustoxoid, monophosphoryl lipid A (MPL) of, for example, E. coli,Salmonella minnesota, Salmonella typhimurium, or Shigella exseri),particulate adjuvants (biodegradable, polymer microspheres),immunostimulatory complexes (ISCOMs)), oil-emulsion and surfactant-basedadjuvants (Freund's incomplete adjuvant (FIA), microfluidized emulsions(MF59, SAF), saponins (QS-21)), synthetic (muramyl peptide derivatives(murabutide, threony-MDP), nonionic block copolymers (L121),polyphosphazene (PCCP), synthetic polynucleotides (poly A:U, poly I:C),thalidomide derivatives (CC-4407/ACTIMID)), RH3-ligand, or polylactideglycolide (PLGA) microspheres, among others. Fragments, homologs,derivatives, and fusions to any of these toxins are also suitable,provided that they retain adjuvant activity. Suitable mutants orvariants of adjuvants are described, e.g., in WO 95/17211 (Arg-7-Lys CTmutant), WO 96/6627 (Arg-192-Gly LT mutant), and WO 95/34323 (Arg-9-Lysand Glu-129-Gly PT mutant). Additional LT mutants that can be used inthe methods and compositions of the invention include, e. g.,Ser-63-Lys, Ala-69-Gly,Glu-110-Asp, and Glu-112-Asp mutants. Othersuitable adjuvants are also well-known in the art.

As an example, metallic salt adjuvants such alum adjuvants arewell-known in the art as providing a safe excipient with adjuvantactivity. The mechanism of action of these adjuvants are thought toinclude the formation of an antigen depot such that antigen may stay atthe site of injection for up to 3 weeks after administration, and alsothe formation of antigen/metallic salt complexes which are more easilytaken up by antigen presenting cells. In addition to aluminium, othermetallic salts have been used to adsorb antigens, including salts ofzinc, calcium, cerium, chromium, iron, and berilium. The hydroxide andphosphate salts of aluminium are the most common. Formulations orcompositions containing aluminium salts, antigen, and an additionalimmunostimulant are known in the art. An example of an immunostimulantis 3-de-O-acylated monophosphoryl lipid A (3D-MPL).

Any of these components may be used alone or in combination with otheragents. For instance, it has been shown that a combination of CD80,ICAM-1 and LFA-3 (“TRICOM”) may potentiate anti-cancer immune responses(Hodge, et al. Cancer Res. 59: 5800-5807 (1999). Other effectivecombinations include, for example, IL-12+GM-CSF (Ahlers, et al. J.Immunol., 158: 3947-3958 (1997); Iwasaki, et al. J. Immunol. 158:4591-4601 (1997)), IL-12+GM-CSF+TNF-α (Ahlers, et al. Int. Immunol. 13:897-908 (2001)), CD80+IL-12 (Fruend, et al. Int. J. Cancer, 85: 508-517(2000); Rao, et al. supra), and CD86+GM-CSF+IL-12 (Iwasaki, supra). Oneof skill in the art would be aware of additional combinations useful incarrying out the present invention. In addition, the skilled artisanwould be aware of additional reagents or methods that may be used tomodulate such mechanisms. These reagents and methods, as well as othersknown by those of skill in the art, may be utilized in practicing thepresent invention.

Other agents that may be utilized in conjunction with the compositionsand methods provided herein include anti-infective agents (e.g.,antibiotics, anti-viral medications). For example, with respect to HIV,agents including, for example, protease inhibitor, an HIV entryinhibitor, a reverse transcriptase inhibitor, and/or an anti-retroviralnucleoside analog. Suitable compounds include, for example, Agenerase(amprenavir), Combivir (Retrovir/Epivir), Crixivan (indinavir), Emtriva(emtricitabine), Epivir (3tc/lamivudine), Epzicom, Fortovase/Invirase(saquinavir), Fuzeon (enfuvirtide), Hivid (ddc/zalcitabine), Kaletra(lopinavir), Lexiva (Fosamprenavir), Norvir (ritonavir), Rescriptor(delavirdine), Retrovir/AZT (zidovudine), Reyatax (atazanavir,BMS-232632), Sustiva (efavirenz), Trizivir(abacavir/zidovudine/lamivudine), Truvada (Emtricitabine/Tenofovir DF),Videx (ddI/didanosine), Videx EC (ddI, didanosine), Viracept(nevirapine), Viread (tenofovir disoproxil fumarate), Zerit(d4T/stavudine), and Ziagen (abacavir) may be utilized. Other suitableagents are known to those of skill in the art. Such agents may either beused prior to, during, or after administration of the compositionsand/or use of the methods described herein.

Other agents that may be utilized in conjunction with the compositionsand methods provided herein include chemotherapeutics and the like(e.g., chemotherapeutic agents, radiation, anti-angiogenic compounds(Sebti, et al. Oncogene 2000 Dec. 27; 19(56):6566-73)). For example, intreating metastatic breast cancer, useful chemotherapeutic agentsinclude cyclophosphamide, doxorubicin, paclitaxel, docetaxel, navelbine,capecitabine, and mitomycin C, among others. Combinationchemotherapeutic regimens have also proven effective includingcyclophosphamide+methotrexate+5-fluorouracil;cyclophosphamide+doxorubicin+5-fluorouracil; or,cyclophosphamide+doxorubicin, for example. Other compounds such asprednisone, a taxane, navelbine, mitomycin C, or vinblastine have beenutlized for various reasons. A majority of breast cancer patients haveestrogen-receptor positive (ER+) tumors and in these patients, endocrinetherapy (i.e., tamoxifen) is preferred over chemotherapy. For suchpatients, tamoxifen or, as a second line therapy, progestins(medroxyprogesterone acetate or megestrol acetate) are preferred.Aromatase inhibitors (i.e., aminoglutethimide and analogs thereof suchas letrozole) decrease the availability of estrogen needed to maintaintumor growth and may be used as second or third line endocrine therapyin certain patients.

Other cancers may require different chemotherapeutic regimens. Forexample, metastatic colorectal cancer is typically treated withCamptosar (irinotecan or CPT-11), 5-fluorouracil or leucovorin, alone orin combination with one another. Proteinase and integrin inhibitors suchas as the MMP inhibitors marimastate (British Biotech), COL-3(Collagenex), Neovastat (Aeterna), AG3340 (Agouron), BMS-275291 (BristolMyers Squibb), CGS 27023A (Novartis) or the integrin inhibitors Vitaxin(Medimmune), or MED1522 (Merck KgaA) may also be suitable for use. Assuch, immunological targeting of immunogenic targets associated withcolorectal cancer could be performed in combination with a treatmentusing those chemotherapeutic agents. Similarly, chemotherapeutic agentsused to treat other types of cancers are well-known in the art and mayalso be suitable for use.

Many anti-angiogenic agents are known in the art may also be used incombination with the recombinant vectors described herein (see, forexample, Timar, et al. 2001. Pathology Oncol. Res., 7(2): 85-94). Suchagents include, for example, physiological agents such as growth factors(i.e., ANG-2, NK1,2,4 (HGF), transforming growth factor beta (TGF-β)),cytokines (i.e., interferons such as IFN-α, -β, -γ, platelet factor 4(PF-4), PR-39), proteases (i.e., cleaved AT-III, collagen XVIII fragment(Endostatin)), HmwKallikrein-d5 plasmin fragment (Angiostatin),prothrombin-F1-2, TSP-1), protease inhibitors (i.e., tissue inhibitor ofmetalloproteases such as TIMP-1, -2, or −3; maspin; plasminogenactivator-inhibitors such as PAI-1; pigment epithelium derived factor(PEDF)), Tumstatin (available through ILEX, Inc.), antibody products(i.e., the collagen-binding antibodies HUIV26, HUI77, XL313; anti-VEGF;anti-integrin (i.e., Vitaxin, (Lxsys))), and glycosidases (i.e.,heparinase-I, -III). “Chemical” or modified physiological agents knownor believed to have anti-angiogenic potential include, for example,vinblastine, taxol, ketoconazole, thalidomide, dolestatin, combrestatinA, rapamycin (Guba, et al. 2002, Nature Med., 8: 128-135), CEP-7055(available from Cephalon, Inc.), flavone acetic acid, Bay 12-9566 (BayerCorp.), AG3340 (Agouron, Inc.), CGS 27023A (Novartis), tetracylcinederivatives (i.e., COL-3 (Collagenix, Inc.)), Neovastat (Aeterna),BMS-275291 (Bristol-Myers Squibb), low dose 5-FU, low dose methotrexate(MTX), irsofladine, radicicol, cyclosporine, captopril, celecoxib,D45152-sulphated polysaccharide, cationic protein (Protamine), cationicpeptide-VEGF, Suramin (polysulphonated napthyl urea), compounds thatinterfere with the function or production of VEGF (i.e., SU5416 orSU6668 (Sugen), PTK787/ZK22584 (Novartis)), Distamycin A, Angiozyme(ribozyme), isoflavinoids, staurosporine derivatives, genistein,EMD121974 (Merck KcgaA), tyrphostins, isoquinolones, retinoic acid,carboxyamidotriazole, TNP-470, octreotide, 2-methoxyestradiol,aminosterols (i.e., squalamine), glutathione analogues (i.e.,N-acteyl-L-cysteine), combretastatin A-4 (Oxigene), Eph receptorblocking agents (Nature, 414:933-938, 2001), Rh-Angiostatin,Rh-Endostatin (WO 01/93897), cyclic-RGD peptide, accutin-disintegrin,benzodiazepenes, humanized anti-avb3 Ab, Rh-PAI-2, amiloride,p-amidobenzamidine, anti-uPA ab, anti-uPAR Ab,L-phanylalanin-N-methylamides (i.e., Batimistat, Marimastat), AG3340,and minocycline. Other suitable agents are known in the art and may besuitable for use.

Administration of a composition of the present invention to a host maybe accomplished using any of a variety of techniques known to those ofskill in the art. The composition(s) may be processed in accordance withconventional methods of pharmacy to produce medicinal agents foradministration to patients, including humans and other mammals (i.e., a“pharmaceutical composition”). The pharmaceutical composition ispreferably made in the form of a dosage unit containing a given amountof DNA, viral vector particles, polypeptide, peptide, or other drugcandidate, for example. A suitable daily dose for a human or othermammal may vary widely depending on the condition of the patient andother factors, but, once again, can be determined using routine methods.The compositions are administered to a patient in a form and amountsufficient to elicit a therapeutic effect. Amounts effective for thisuse will depend on various factors, including for example, theparticular composition of the vaccine regimen administered, the mannerof administration, the stage and severity of the disease, the generalstate of health of the patient, and the judgment of the prescribingphysician. The dosage regimen for immunizing a host or otherwisetreating a disorder or a disease with a composition of this invention isbased on a variety of factors, including the type of disease, the age,weight, sex, medical condition of the patient, the severity of thecondition, the route of administration, and the particular compoundemployed. Thus, the dosage regimen may vary widely, but can bedetermined routinely using standard methods.

In general, recombinant viruses may be administered in compositions inan amount of about 10⁴ to about 10⁹ pfu per inoculation; often about 10⁴pfu to about 10⁶ pfu, or as shown in the Examples, 10⁷ to 10³ pfu.Higher dosages such as about 10⁴ pfu to about 10¹⁰ pfu, e.g., about 10⁵pfu to about 10⁹ pfu, or about 10⁶ pfu to about 10⁸ pfu, or about 10⁷pfu can also be employed. Another measure commonly used is DICC₅₀;suitable DICC₅₀ ranges for administration include about 10¹, about 10²,about 10³, about 10⁴, about 10⁵, about 10⁶, about 10⁷, about 10⁸, about10⁹, about 10¹⁰ DICC₅₀. Ordinarily, suitable quantities of plasmid ornaked DNA are about 1 μg to about 100 mg, about 1 mg, about 2 mg, butlower levels such as 0.1 to 1 mg or 1-10 μg may be employed. Actualdosages of such compositions can be readily determined by one ofordinary skill in the field of vaccine technology.

The pharmaceutical composition may be administered orally, parentally,by inhalation spray, rectally, intranodally, or topically in dosage unitformulations containing conventional pharmaceutically acceptablecarriers, adjuvants, and vehicles. The term “pharmaceutically acceptablecarrier” or “physiologically acceptable carrier” as used herein refersto one or more formulation materials suitable for accomplishing orenhancing the delivery of a nucleic acid, polypeptide, or peptide as apharmaceutical composition. A “pharmaceutical composition” is acomposition comprising a therapeutically effective amount of a nucleicacid or polypeptide. The terms “effective amount” and “therapeuticallyeffective amount” each refer to the amount of a nucleic acid orpolypeptide used to observe the desired therapeutic effect (e.g., induceor enhance and immune response).

Injectable preparations, such as sterile injectable aqueous oroleaginous suspensions, may be formulated according to known methodsusing suitable dispersing or wetting agents and suspending agents. Theinjectable preparation may also be a sterile injectable solution orsuspension in a non-toxic parenterally acceptable diluent or solvent.Suitable vehicles and solvents that may be employed are water, Ringer'ssolution, and isotonic sodium chloride solution, among others. Forinstance, a viral vector such as a poxvirus may be prepared in 0.4% NaClor a Tris-HCl buffer, with or without a suitable stabilizer such aslactoglutamate, and with or without freeze drying medium. In addition,sterile, fixed oils are conventionally employed as a solvent orsuspending medium. For this purpose, any bland fixed oil may beemployed, including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid find use in the preparation of injectables.

Pharmaceutical compositions may take any of several forms and may beadministered by any of several routes. The compositions are administeredvia a parenteral route (e.g., intradermal, intramuscular, subcutaneous,skin scarification) to induce an immune response in the host.Alternatively, the composition may be administered directly into a lymphnode (intranodal) or tumor mass (i.e., intratumoral administration).Preferred embodiments of administratable compositions include, forexample, nucleic acids, viral particles, or polypeptides in liquidpreparations such as suspensions, syrups, or elixirs. Preferredinjectable preparations include, for example, nucleic acids orpolypeptides suitable for parental, subcutaneous, intradermal,intramuscular or intravenous administration such as sterile suspensionsor emulsions. For example, a naked DNA molecule and/or recombinantpoxvirus may separately or together be in admixture with a suitablecarrier, diluent, or excipient such as sterile water, physiologicalsaline, glucose or the like. The composition may also be provided inlyophilized form for reconstituting, for instance, in isotonic aqueous,saline buffer. In addition, the compositions can be co-administered orsequentially administered with one another, other antiviral compounds,other anti-cancer compounds and/or compounds that reduce or alleviateill effects of such agents.

As previously mentioned, while the compositions described herein may beadministered as the sole active pharmaceutical agent, they can also beused in combination with one or more other compositions or agents (i.e.,other immunogens, co-stimulatory molecules, adjuvants). Whenadministered as a combination, the individual components can beformulated as separate compositions administered at the same time ordifferent times, or the components can be combined as a singlecomposition. In one embodiment, a method of administering to a host afirst form of an immunogen and subsequently administering a second formof the immunogen, wherein the first and second forms are different, andwherein administration of the first form prior to administration of thesecond form enhances the immune response resulting from administrationof the second form relative to administration of the second form alone,is provided. Also provided are compositions for administration to thehost. For example, a two-part immunological composition where the firstpart of the composition comprises a first form of an immunogen and thesecond part comprises a second form of the immunogen, wherein the firstand second parts are administered separately from one another such thatadministration of the first form enhances the immune response againstthe second form relative to administration of the second form alone, isprovided. The immunogens, which may be the same or different, arepreferably derived from the infectious agent or other source ofimmunogens. The multiple immunogens may be administered together orseparately, as a single or multiple compositions, or in single ormultiple recombinant vectors.

A kit comprising a composition of the present invention is alsoprovided. The kit can include a separate container containing a suitablecarrier, diluent or excipient. The kit may also include additionalcomponents for simultaneous or sequential-administration. In oneembodiment, such a kit may include a first form of an immunogen and asecond form of the immunogen. Additionally, the kit can includeinstructions for mixing or combining ingredients and/or administration.A kit may provide reagents for performing screening assays, such as oneor more PCR primers, hybridization probes, and/or biochips, for example.

All references cited within this application are incorporated byreference. A better understanding of the present invention and of itsmany advantages will be had from the following examples, given by way ofillustration.

EXAMPLES Example 1 NYVAC-HIV C Vector

The recombinant vectors DNA C and NYVAC-HIV C expressed HIV genesderived from the Chinese R5 clade C virus (97CN54; Su, et al. J. Virol.2000. 74: 11367-76; WO 01/36614; Gomez et al., Vaccine, Vol. 25, pp.1969-1992 (2007)). This clone has been shown to be representative ofclade C strains circulating in China and India. All HIV genes have beenoptimised for codon usage since it has recently been shown thathumanization of synthetic HIV gene codons allowed for an enhanced andREV/RRE- independent expression of env and gag-pol genes in mammaliancells. Genes were optimized for both safety and translation efficiency.The env gene has been designed to express the secreted gp120 form of theenvelope proteins and contain an optimal synthetic leader sequence forenhanced expression. The gag, pol and nef genes were fused to express aGAG-POL-NEF polyprotein. An artificial −1 frameshift introduced in thenatural slippery sequence of the p7-p6 gene junction results in anin-frame GAG-POL-NEF fusion protein due to the absence of ribosomalframeshift. An N-terminal Gly→Ala substitution in gag prevents theformation and release of virus-like particles from transfected cells.This strategy allows for an equimolar production of GAG, POL and NEFproteins and an enhanced MEW Class-I restricted presentation of theirCTL epitopes. For safety and regulatory reason, the packaging signalsequence has been removed; the integrase gene deleted; and the reversetranscriptase gene disrupted by insertion of a scrambled nef gene at the3′ end of the DNA sequence coding for the RT active site known to beassociated with an immunodominant CTL epitope. The nef gene has beendislocated by fusing its 5′ half to its 3′ half without losing itsimmunodominant CTL epitopes.

A. NYVAC-HIV-C(vP2010)

1. Donor Plasmid pMA60gp120C/Gagpolnef-C-14.

Donor plasmid pMA60gp120C/GAG-POL-NEF-C-14 was constructed forengineering of NYVAC or MVA expressing HIV-1 clade C gp120 envelope andGAG-POL-NEF proteins. The plasmid is a pUC derivative containing TK leftand right flanking sequences in pUC cloning sites. Between two flankingsequences two synthetic early/late (E/L) promoters in a back to backorientation individually drive codon-optimized clade C gp120 gene andgag-pol-nef gene. The locations of the TK flanking sequences, E/Lpromoters, transcriptional termination signal, gp120 and gag-pol-nefgenes as described in Table 2 below:

TABLE 2 pMA60gp120C/gagpolnef-C-14 Left flanking sequence Nt. 1609-2110(complementary) Right flanking sequence Nt. 4752-5433 (complementary)E/L promoter for gp120 Nt. 12-51 Gp120 gene (ATG-TGA) Nt 61-1557Terminal signal for gp120 Nt. 1586-1592 E/L promoter for gagpolnef Nt.9794-9833 (complementary) gagpolnef gene (ATG-TAA) Nt. 5531-9784(complementary) Terminal signal for gagpolnef Nt. 5422-5416(complementary)2. Construction of pMA60gp120C/Gagpolnef-C-14 DNA Origin:a. pMA60: This plasmid is a pUC derivative containing TK right and leftflanking sequences in pUC cloning sites. Between the two flankingsequences there is a synthetic E/L promoter. The left flanking sequenceis located at 37-550 and right flanking sequence is at 610-1329. The E/Lpromoter (AAAATTGAAATTTTATTTTTTTTTTTTGGAATATAAATA; SEQ ID NO. 60) islocated at 680-569.b. pCR-Script clade C-syngp120: The plasmid contained a codon-optimizedclade C HIV-1 gp120 gene. The gp120 gene is located at nucleotides1-1497 (ATG to TAA).c. pCRs-cript clade C-syngagpolnef: The plasmid containing acodon-optimized clade C HIV-1 gagpolnef gene was provided by Hans Wolfand Walf Wagner (Regensburg University, Germany). The gagpolnef gene waslocated between nucleotides 1-4473 (ATG to TAA).d. pSE1379.7: The plasmid is a Bluescript derivative containing asynthetic E/L promoter. The E/L promoter is located at nucleotides1007-968.3. Construction of pMA60 gp120C/Gagpolnef-C-14:a. Construction of pMA60-T5NT-24: pMA60 has a synthetic E/L promoter buthas no transcriptional termination signal for the promoter. To insert aterminal signal TSNT for the promoter, a DNA fragment composed of a pairof oligonucleotides, 5′-CCGGAATTTTTATT-3′(7291) (SEQ ID NO.61)/3′-TTAAAAATAAGGCC-5′ (7292) (SEQ ID NO. 62), was inserted into Xma Isite on pMA60. The resulted plasmid was designated pMA60-T5NT-24.b. Construction of pMA60gp120C-10: To generate a clade C gp120 genewithout extra sequence between promoter and start codon ATG a KpnI-KpnIfragment (nt. 4430-1527) containing the gp120 gene was isolated frompCR-Script clade C-syngp120 and used as template in a PCR. In the PCR,primers 7490/7491 (7490: 5′-TTGAATTCTCGAGCATGGACAGGGCCAAGCTGCTGCTGCTGCTG(SEQ ID NO. 63) and 7491: 5′-TGCTGCTCACGTTCCTGCACTCCAGGGT (SEQ ID NO.64)) were used to amplify a ˜370 bp 5′-gp120 fragment. The fragment wascut with EcoRI and AatII generating an EcoRI-AatII fragment (˜300 bp).The EcoRI-AatII fragment was used to replace a corresponding EcoRI-AatII fragment (nt. 4432-293) on pCR-Script clade C-syngp120 resulting in aplasmid pCR-Script clade Cgp120-PCR-19. A XhoI-XhoI fragment containinga gp120 gene was isolated from pCR-Script cladeCgp120-PCR-19 and clonedinto XhoI site on pMA60-T5NT-24 generating pMA60gp120C-10.c. Construction of pMA60gp120C/gagpolnef-C-14: To create a clade Cgagpolnef gene without extra sequence between promoter and stat codon ofthe gene a KpnI-KpnI (nt 7313-4352) fragment containing the gagpolnefgene was isolated from pCRscript-Syngagpolnef and used as template in aPCR reaction. The primers were oligonucleotides (7618: 5′TTTCTCGAGCATGGCCGCCAGGGCCAGCATCCTGAGG (SEQ ID NO. 65)/7619:5′-ATCTGCTCCTGCAGGTTGCTGGTGGT (SEQ ID NO. 66). A fragment (˜740 bp)amplified in the PCR was cloned into Sma I site on pUC18 resulting in aplasmid designated pATGgpn-740. The ˜740 bp fragment in pATGgpn-740 wasconfirmed by DNA sequencing. The pATGgpn-740 was cut with XhoI and StuIgenerating an XhoI-StuI fragment (˜480 bp). In addition,pCRScript-syngagpolnef was cut with StuI and KpnI generating a StuI-KpnIfragment (nt. 479-4325). Meanwhile pSE1379.7, a Bluescript derivativecontaining an E/L promoter, was linealized with XhoI and KpnI generatingan XhoI-KpnI receptor fragment (˜3 kb). The two fragments (XhoI-Stu Iand StuI-KpnI) and the receptor fragment (XhoI-KpnI) were ligatedtogether generating a plasmid pATGgagpolnef-C-2. Finally, thepATG-gagpolnef-C-2 was cut with SalI generating a SalI-SalI fragmentthat contained an E/L-gagpolnef cassette. The SalI-SalI fragment wascloned into a SalI site on pMA60gp120C-10 generatingpMA60gp120C/gagpolnef-C-14.4. Generation of NYVAC-HIV-C Recombinant (vP2010; “NYVAC-C”)

The IVR was performed by transfection of 1°CEF cells (Merial product)with pMA60gp120C/gagpolenef C-14 using calcium phosphate method andsimultaneously infection of the cells with NYVAC as rescue virus at MOIof 10. After ˜14 hr, the transfected-infected cells were harvested,sonicated and used for recombinant virus screening. Recombinant plaqueswere screened based on plaque lift hybridization method. A 1.5 kb cladeC gp120 gene that was labeled with p32 according to a random primerlabeling kit protocol (Promega) was used as probe. In the first roundscreening, ˜11700 plaques were screened and three positive clonesdesignated vP2010-1,vP 2010-2, vP2010-3, were obtained. After sequentialfour rounds of plaque purification, recombinants designatedvP2010-1-2-1-1, vP2010-1-2-2-1, vP2010-1-4-1-1, vP2010-1-4-1-2 andvP2010-1-4-2-1 were generated and confirmed by hybridization as 100%positive using the gp120 probe. P2 stocks of these recombinants wereprepared. A P3 (roller bottle) stock with a titer 1.2×10⁹ was prepared.

Example 2 Modified Expression Vectors A. Immunomodulatory Vectors

The plasmid backbone used for the generation of the different plasmidtransfer vectors is termed pGem-Red-GFP wm (FIG. 1) This plasmid,derived from pGem-7Zf(−) (Promega Corp.), contains two differentfluorescent proteins (Red2 and rsGFP), each under the control of thevaccinia virus synthetic early/late promoter. The plasmid transfervectors listed in Table 3 were generated by the sequential cloning ofthe recombination flanking sequences of the specific genes to bedeleted.

TABLE 3 Plasmid transfer vector Deleted Gene Recombinant ViruspGem-RG-B8R wm B8R NYVAC-C-ΔB8R NYVAC-C-ΔB8R/B19R pGem-RG-B19R wm B19RNYVAC-C-ΔB19R NYVAC-C-ΔB8R/B19R

2. NYVAC-C-ΔB8R Recombinant Vectors

The plasmid transfer vector pGem-RG-B8R wm, used for the construction ofthe recombinant virus “NYVAC-C-ΔB8R”, having the B8R open reading frame(e.g, SEQ ID NO. 2 encoding SEQ ID NO. 1) deleted, was obtained bysequential cloning of five DNA fragments containing dsRed2 and rsGFPgenes and B8R recombination flanking sequences into the plasmidpGem-7Zf(−) (Promega). The dsRed2 gene under the control of thesynthetic early/late promoter was amplified by PCR from plasmidpG-dsRed2 with oligonucleotides Red2-B (5′-GAACTAGGATCCTAA CTCGAGAAA-3′;SEQ ID NO. 67) (Bam HI site underlined) andRed2-N(5′-ATTAGTATGCATTTATTTATTTAGG-3′; SEQ ID NO. 68) (Nsi I siteunderlined) (785 bp), digested with Bam HI and Nsi I and inserted intothe Bam HI/Nsi I-digested pGem-7Zf(−) to generate pGem-Red wm (3740 bp).The rsGFP gene under the control of the synthetic early/late promoterwas amplified by PCR from plasmid pG-dsRed2 with oligonucleotides GFP-X(5′-CGTTGGTCTAGAGAGAAAAATTG-3′; SEQ ID NO. 69) (Xba I site underlined)and GFP-E (5′-CTATAGAATTCTCAAGCTATGC-3′; SEQ ID NO. 70) (Eco RI siteunderlined) (832 bp), digested with Xba I and Eco RI and inserted intoplasmid pGem-Red wm previously digested with Xba I and Eco RI to obtainpGem-Red-GFP wm (4540 bp).

NYVAC genome (FIG. 2) was used as the template to amplify the left flankof B8R gene (358 bp) with oligonucleotides LFB8R-AatII-F(5′-TTTTTTGACGTCATTGACTCGTCTACTATTC-3′; SEQ ID NO. 71) (Aat II siteunderlined) and LFB8R-XbaI-R (5″-TTTTTTTCTAGATGG TGTTGTTTGTTATTTG-3′;SEQ ID NO. 72) (Xba I site underlined). This left flank was digestedwith Aat II and Xba I and cloned into plasmid pGem-Red-GFP wm previouslydigested with the same restriction enzymes to generate pGem-RG-LFsB8R wm(4865 bp). The repeated left flank of B8R gene (358 bp) was amplified byPCR from NYVAC genome with oligonucleotides LFB8R′-EcoRI-F(5′-TTTTTTGAATTCATTGACTCGTCTACTATTC-3′; SEQ ID NO. 73) (Eco RI siteunderlined) and LFB8W-ClaI-R (5″-TTTTTTATCGATTGGTGTTGTTTGTTATTTG-3′; SEQID NO. 74) (Cla I site underlined), digested with Eco RI and Cla I andinserted into the Eco RI/Cla I-digested pGem-RG-LFsB8R wm to generatepGem-RG-LFdB8R wm (5182 bp). The right flank of B8R gene (367 bp) wasamplified by PCR from NYVAC genome with oligonucleotides RFB8R-ClaI-F(5′-TTTTTTATCGATCTAATTT TTATTAATGATAC-3′; SEQ ID NO. 75) (Cla I siteunderlined) and RFB8R-BamHI-R (5′-TTTTTTGGATCCAAACAGCGGACACATTGC-3′; SEQID NO. 76) (Barn HI site underlined), digested with Cla I and Barn HIand inserted into the Cla I/Barn HI-digested pGem-RG-LFdB8R wm. Theresulting plasmid pGem-RG-B8R wm (5519 bp; FIGS. 3A and 3B) wasconfirmed by DNA sequence analysis and directs the deletion of B8R genefrom NYVAC-C genome.

This deletion mutant NYVAC-C-ΔB8R was constructed by transient dominantselection using dsRed2 and rsGFP genes as the transiently selectablemarkers. 3×10⁶ BSC-40 cells were infected with 0.01 PFU/cell of NYVAC-Cand transfected 1 h later with 6 μg DNA of plasmid pGem-RG-B8R wm usingLipofectamine (Invitrogen, San Diego, Calif.) according to themanufacturer's recommendations. After 48 h post-infection, the cellswere harvested, lysed by freeze-thaw cycling, sonicated and used forrecombinant virus screening. The deletion mutant was selected fromprogeny virus by consecutive rounds of plaque purification in BSC-40cells during which plaques were screened for Red2/GFP fluorescence. Inthe first two passages, viruses from selected plaques expressed bothfluorescent proteins, in the next two passages viral progeny fromselected plaques expressed only one fluorescent marker (Red2 or GFP) andin the last two passages (six passages in total) viruses from selectedplaques do not express any marker due to the loss of the fluorescentmarker. The deletion mutant was detected by PCR amplifying the B8Rlocus.

The resulting NYVAC-C-ΔB8R positive virus plaques were grown in BSC-40cells, and further passage twice in primary CEF cells. A P-2 stock wasprepared in CEF and used for the propagation of the virus in CEF,followed by virus purification through two 36% (w/v) sucrose cushions in10 mM Tris-HCl pH 9, and titrated by plaque assay in BSC-40 cells. Thepurified grown stock of virus was referred as P-3.

To test the purity of the deletion mutant NYVAC-C-4B8R, viral DNA wasextracted by the method of SDS-Proteinase K-Phenol from BSC-40 cellsmock-infected or infected at 5 PFU/cell with NYVAC-C-ΔB8R. PrimersLFB8R-AatII-F and LFB8R-BamHI-R spanning B8R flanking regions were usedfor PCR analysis of B8R locus. The amplification reactions were carriedout with Platinum Taq DNA polymerase (Invitrogen, San Diego, Calif.)(results shown in FIG. 4).

To test the correct expression of HIV proteins gp120 and GPN fromNYVAC-C-ΔB8R, monolayers of BSC-40 cells were mock-infected or infectedat 5 PFU/cell with NYVAC wt, NYVAC-C, NYVAC-C-4B8R. At 48 hpost-infection, cells were lysed in Laemmli buffer, cells extractsfractionated by 8% SDS-PAGE and analyzed by Western blot using rabbitpolyclonal anti-gp120 antibody (Centro Nacional de Biotecnologia;diluted 1:3000) or polyclonal anti-gag p24 serum (ARP 432, NIBSC,Centralised Facility for AIDS reagent, UK; diluted 1:1000) followed byanti-rabbit-HRP (Sigma; diluted 1:5000) to evaluate the expression ofgp120 and GPN proteins (FIG. 5).

To evaluate the stability of HIV proteins expressed by NYVAC-C-4B8R,monolayers of BSC-40 cells grown in 6 well-plates were infected withserial dilutions of NYVAC wt, NYVAC-C, NYVAC-C-4B8R produced after 12successive passages. At 42 h post-infection, the viruses were titratedby plaque immunostaining assay using rabbit polyclonal antibody againstvaccinia virus strain WR (Centro Nacional de Biotecnologia; diluted1:1000) or rabbit polyclonal anti-gp120 antibody (Centro Nacional deBiotecnologia; diluted 1:250) followed by anti-rabbit-HRP (Sigma;diluted 1:1000) (FIG. 6). All recombinant viruses showed similarimmunoreactivity to both anti-WR and anti-gp120.

To determine virus-growth profiles, monolayers of CEF cells grown in12-well tissue culture plates were infected in duplicate at 0.01PFU/cell with NYVAC wt, NYVAC-C, NYVAC-C-4B8R. Following virusadsorption for 60 min at 37° C., the inoculum was removed. The infectedcells were washed once with DMEM without serum and incubated with freshDMEM containing 2% FCS at 37° C. in a 5% CO₂ atmosphere. At differenttimes post-infection (0, 24, 48 and 72 hours), cells were removed byscraping (lysates at 5×10⁵ cells/ml), freeze-thawed three times andbriefly sonicated. Virus titers in cell lysates were determined bycrystal violet staining in BSC-40 cells (FIG. 7).

2. NYVAC-C-ΔB19R Recombinant Vectors

The plasmid transfer vector pGem-RG-B19R wm, used for the constructionof the recombinant viruse “NYVAC-C-ΔB19R”, having the B19R open readingframe (e.g, SEQ ID NO. 4 encoding SEQ ID NO. 3), was obtained by thesequential cloning of B19R recombination flanking sequences into theplasmid pGem-Red-GFP wm (previously described). NYVAC genome (FIG. 2)was used as the template to amplify the left flank of B19R gene (364 bp)with oligonucleotides LFB19R-AatII-F(5′-TTTTTTGACGTCGAGAAAGTTAAGAAGATAC-3′; SEQ ID NO. 77) (Aat II siteunderlined) and LFB19R-XbaI-R (5′-TTTTTTTCTAGATCTTTATTATACGGCACTAA-3′;SEQ ID NO. 78) (Xba I site underlined). This left flank was digestedwith Aat II and Xba I and cloned into plasmid pGem-Red-GFP wm previouslydigested with the same restriction enzymes to generate pGem-RG-LFsB19Rwm (4871 bp). The repeated left flank of B19R gene (364 bp) wasamplified by PCR from NYVAC genome with oligonucleotides LFB19R′-EcoRI-F(5′-TTTTTTGAATTCGAGAAAGTTAAGA AGATAC-3′; SEQ ID NO. 79) (Eco RI siteunderlined) and LFB19W-ClaI-R (5′-TTTTTTATCGAT TCTTTATTATACGGCACTAA-3′;SEQ ID NO. 80) (Cla I site underlined), digested with Eco RI and Cla Iand inserted into the Eco RI/Cla I-digested pGem-RG-LFsB19R wm togenerate pGem-RG-LFdB19R wm (5194 bp). The right flank of B19R gene (381bp) was amplified by PCR from NYVAC genome with oligonucleotidesRFB19R-ClaI-F (5′-TTTTTTATCGATATATACAATGCATTTTTATATAC-3′; SEQ ID NO. 81)(Cla I site underlined) and RFB19R-BamHI-R (5′-TTTTTTGGATCCAGTTCTATCATAATCATC-3′; SEQ ID NO. 82) (Bam HI site underlined), digested withCla I and Bam HI and inserted into the Cla I/Bam HI-digestedpGem-RG-LFdB19R wm. The resulting plasmid pGem-RG-B19R wm (5545 bp;FIGS. 8A and 8B) was confirmed by DNA sequence analysis and directs thedeletion of B19R gene from NYVAC-C genomes.

This deletion mutant NYVAC-C-ΔB19R was constructed by transient dominantselection using dsRed2 and rsGFP genes as the selectable markers. 3×10⁶BSC-40 cells were infected with 0.01 PFU/cell of NYVAC-C and transfected1 h later with 6 μg DNA of plasmid pGem-RG-B19R wm using Lipofectamine(Invitrogen, San Diego, Calif.). After 48 h post-infection, the cellswere harvested, lysed by freeze-thaw cycling, sonicated and used forrecombinant virus screening. The deletion mutant was selected fromprogeny virus by consecutive rounds of plaque purification in BSC-40cells during which plaques were screened for Red2/GFP fluorescence. Inthe first two passages, viruses from selected plaques expressed bothfluorescent proteins. In the next two passages, viral progeny fromselected plaques expressed only one fluorescent marker. In the last twopassages, viruses from selected plaques do not express any marker due tothe loss of the fluorescent marker. The deletion mutant was detected byPCR amplifying the B19R locus.

The resulting NYVAC-C-4B19R positive virus plaques were grown in BSC-40cells, and further passage twice in primary CEF cells. A P-2 stock wasprepared in CEF and used for the propagation of the virus in CEF,followed by virus purification through two 36% (w/v) sucrose cushions in10 mM Tris-HCl pH 9, and titrated by plaque assay in BSC-40 cells. Thepurified grown stock of virus was referred as P-3.

To test the purity of the deletion mutant NYVAC-C-4B19R, viral DNA wasextracted by the method of SDS-Proteinase K-Phenol from BSC-40 cellsmock-infected or infected at 5 PFU/cell with NYVAC-C-4B19R. PrimersLFB19R-AatII-F and LFB19R-BamHI-R spanning B19R flanking regions wereused for PCR analysis of B19R locus. The amplification reactions werecarried out with Platinum Taq DNA polymerase (Invitrogen, San Diego,Calif.) (FIG. 4).

To test the correct expression of HIV proteins gp120 and GPN fromNYVAC-C-ΔB19R, monolayers of BSC-40 cells were mock-infected or infectedat 5 PFU/cell with NYVAC wt, NYVAC-C, NYVAC-C-ΔB19R. At 48 hpost-infection, cells were lysed in Laemmli buffer, cells extractsfractionated by 8% SDS-PAGE and analyzed by Western blot using rabbitpolyclonal anti-gp120 antibody (Centro Nacional de Biotecnologia;diluted 1:3000) or polyclonal anti-gag p24 serum (ARP 432, NIBSC,Centralised Facility for AIDS reagent, UK; diluted 1:1000) followed byanti-rabbit-HRP (Sigma; diluted 1:5000) to evaluate the expression ofgp120 and GPN proteins (FIG. 5).

To evaluate the stability of HIV proteins expressed by NYVAC-C-4B19R,monolayers of BSC-40 cells grown in 6 well-plates were infected withserial dilutions of NYVAC wt, NYVAC-C, NYVAC-C-4B19R produced after 12successive passages. At 42 h post-infection, the viruses were titratedby plaque immunostaining assay using rabbit polyclonal antibody againstvaccinia virus strain WR (Centro Nacional de Biotecnologia; diluted1:1000) or rabbit polyclonal anti-gp120 antibody (Centro Nacional deBiotecnologia; diluted 1:250) followed by anti-rabbit-HRP (Sigma;diluted 1:1000) (FIG. 6). All recombinant viruses showed similarimmunoreactivity to both anti-WR and anti-gp120.

To determine virus-growth profiles, monolayers of CEF cells grown in12-well tissue culture plates were infected in duplicate at 0.01PFU/cell with NYVAC wt, NYVAC-C, NYVAC-C-ΔB19R. Following virusadsorption for 60 min at 37° C., the inoculum was removed. The infectedcells were washed once with DMEM without serum and incubated with freshDMEM containing 2% FCS at 37° C. in a 5% CO₂ atmosphere. At differenttimes post-infection (0, 24, 48 and 72 hours), cells were removed byscraping (lysates at 5×10⁵ cells/ml), freeze-thawed three times andbriefly sonicated. Virus titers in cell lysates were determined bycrystal violet staining in BSC-40 cells (FIG. 7).

4. NYVAC-C-ΔB8R/B19R Recombinant Vectors

The plasmid transfer vector pGem-RG-B19R wm, used for the constructionof the recombinant virus “NYVAC-C-ΔB8R/B19R”, having the B8R openreading frame (e.g, SEQ ID NO. 2 encoding SEQ ID NO. 1) and the B19Ropen reading frame (e.g, SEQ ID NO. 4 encoding SEQ ID NO. 3) deletedfrom the NYVAC genome, was obtained by the sequential cloning of B19Rrecombination flanking sequences into the plasmid pGem-Red-GFP wm(previously described). NYVAC genome (FIG. 2) was used as the templateto amplify the left flank of B19R gene (364 bp) with oligonucleotidesLFB19R-AatII-F (5′-TTTTTTGACGTCGAGAAAGTTAAGAAGATAC-3′; SEQ ID NO. 77)(Aat II site underlined) and LFB19R-XbaI-R(5′-TTTTTTTCTAGATCTTTATTATACGGCACTAA-3′; SEQ ID NO. 78) (Xba I siteunderlined). This left flank was digested with Aat II and Xba I andcloned into plasmid pGem-Red-GFP wm previously digested with the samerestriction enzymes to generate pGem-RG-LFsB19R wm (4871 bp). Therepeated left flank of B19R gene (364 bp) was amplified by PCR fromNYVAC genome with oligonucleotides LFB19W-EcoRI-F(5′-TTTTTTGAATTCGAGAAAGTTAAGAAGATAC-3′; SEQ ID NO. 79) (Eco RI siteunderlined) and LFB19W-ClaI-R (5′-TTTTTTATCGATTCTTTATTATACGGCACTAA-3′;SEQ ID NO. 80) (Cla I site underlined), digested with Eco RI and Cla Iand inserted into the Eco RI/Cla I-digested pGem-RG-LFsB19R wm togenerate pGem-RG-LFdB19R wm (5194 bp). The right flank of B19R gene (381bp) was amplified by PCR from NYVAC genome with oligonucleotidesRFB19R-ClaI-F (5″-TTTTTTATCGATATATACAATGCATTTTTATATAC-3″) (Cla I siteunderlined; SEQ ID NO. 81) and RFB19R-BamHI-R (5′-TTTTTTGGATCCAGTTCTATCATAATCATC-3′; SEQ ID NO. 82) (Bam HI site underlined), digested withCla I and Bam HI and inserted into the Cla I/Bam HI-digestedpGem-RG-LFdB19R win. The resulting plasmid pGem-RG-B19R win (5545 bp;FIG. 8A and FIG. 8B) was confirmed by DNA sequence analysis and directsthe deletion of B19R gene from NYVAC-C-ΔB8R genomes.

This deletion mutant, NYVAC-C-ΔB8R/B19R, was constructed by transientdominant selection using dsRed2 and rsGFP genes as the selectablemarkers. 3×10⁶ BSC-40 cells were infected with 0.01 PFU/cell ofNYVAC-C-4B8R and transfected 1 h later with 6 μg DNA of plasmidpGem-RG-B19R win using Lipofectamine (Invitrogen, San Diego, Calif.).After 48 h post-infection, the cells were harvested, lysed byfreeze-thaw cycling, sonicated and used for recombinant virus screening.Deletion mutant was selected from progeny virus by consecutive rounds ofplaque purification in BSC-40 cells during which plaques were screenedfor Red2/GFP fluorescence. In the first two passages viruses fromselected plaques expressed both fluorescent proteins. In the next twopassages, viral progeny from selected plaques expressed only onefluorescent marker. In the last two passages, viruses from selectedplaques do not express any marker due to the loss of the fluorescentmarker. The deletion mutant was detected by PCR amplifying the B19Rlocus.

The resulting NYVAC-C-ΔB8P/B19R positive virus plaques were grown inBSC-40 cells, and further passaged twice in primary CEF cells. A P-2stock was prepared in CEF and used for the propagation of the virus inCEF, followed by virus purification through two 36% (w/v) sucrosecushions in 10 mM Tris-HCl pH 9, and titrated by plaque assay in BSC-40cells. The purified grown stock of virus was referred as P-3.

To test the purity of the deletion mutant NYVAC-C-ΔB8P/B19R, viral DNAwas extracted by the method of SDS-Proteinase K-Phenol from BSC-40 cellsmock-infected or infected at 5 PFU/cell with NYVAC-C-ΔB8P/B19R. PrimersLFB8R-AatII-F and LFB8R-BamHI-R spanning B8R flanking regions were usedfor PCR analysis of B8R locus. Primers LFB19R-AatII-F and LFB19R-BamHI-Rspanning B19R flanking regions were used for PCR analysis of B19R locusThe amplification reactions were carried out with Platinum Taq DNApolymerase (Invitrogen, San Diego, Calif.) (FIG. 4).

To test the correct expression of HIV proteins gp120 and GPN fromNYVAC-C-ΔB8R/B19R, monolayers of BSC-40 cells were mock-infected orinfected at 5 PFU/cell with NYVAC wt, NYVAC-C, NYVAC-C-ΔB8P/B19R. At 48h post-infection, cells were lysed in Laemmli buffer, cells extractsfractionated by 8% SDS-PAGE and analyzed by Western blot using rabbitpolyclonal anti-gp120 antibody (Centro Nacional de Biotecnologia;diluted 1:3000) or polyclonal anti-gag p24 serum (ARP 432, NIBSC,Centralised Facility for AIDS reagent, UK; diluted 1:1000) followed byanti-rabbit-HRP (Sigma; diluted 1:5000) to evaluate the expression ofgp120 and GPN proteins (FIG. 5).

To evaluate the stability of HIV proteins expressed byNYVAC-C-ΔB8R/B19R, monolayers of BSC-40 cells grown in 6 well-plateswere infected with serial dilutions of NYVAC wt, NYVAC-C,NYVAC-C-ΔB8R/B19R produced after 12 successive passages. At 42 hpost-infection, the viruses were titrated by plaque immunostaining assayusing rabbit polyclonal antibody against vaccinia virus strain WR(Centro Nacional de Biotecnologia; diluted 1:1000) or rabbit polyclonalanti-gp120 antibody (Centro Nacional de Biotecnologia; diluted 1:250)followed by anti-rabbit-HRP (Sigma; diluted 1:1000) (FIG. 6). Allrecombinant viruses showed similar immunoreactivity to both anti-WR andanti-gp120.

To determine virus-growth profiles, monolayers of CEF cells grown in12-well tissue culture plates were infected in duplicate at 0.01PFU/cell with NYVAC wt, NYVAC-C, NYVAC-C-ΔB8R/B19R. Following virusadsorption for 60 min at 37° C., the inoculum was removed. The infectedcells were washed once with DMEM without serum and incubated with freshDMEM containing 2% FCS at 37° C. in a 5% CO₂ atmosphere. At differenttimes post-infection (0, 24, 48 and 72 hours), cells were removed byscraping (lysates at 5×10⁵ cells/ml), freeze-thawed three times andbriefly sonicated. Virus titers in cell lysates were determined bycrystal violet staining in BSC-40 cells (FIG. 7).

B. Replication Competent NYVAC

The development of attenuated, replication competent strains of vacciniavirus that induce a potent immune response is described below. It isknown in the art that replication-defective strains of vaccinia virusoften do not induce a sufficiently potent immune response to betherapeutically useful. This may be due to the limitation in replicationand the failure of most strains of vaccinia virus to inducepro-inflammatory signal transduction and pro-inflammatory geneexpression. The recombinant vectors described herein have been developedto provide a solution to these problems. As shown below,replication-competent, attenuated strains of vaccinia virus inducepotent pro-inflammatory signal transduction and pro-inflammatory geneexpression.

1. NYVAC-C-KC and NYVAC-C+12 Recombinant Vectors

During construction of NYVAC, a non-essential region of the vacciniavirus genome containing twelve genes flanked by the K1L and C7L hostrange genes was deleted. Deletion of genes in this region renders NYVACreplication-defective in human cells. As shown below, replicationcompetence of NYVAC was restored by re-insertion of the two host rangegenes C7L and K1L into NYVAC (NYVAC-KC) or NYVAC-C (NYVAC-C-KC), orre-insertion of the entire host range region (C1L (e.g., SEQ ID NOS. 5,6), C2L (e.g., SEQ ID NOS. 7, 8), C3L (e.g., SEQ ID NOS. 9, 10), C4L(SEQ ID NOS. 11, 12), C5L (e.g., SEQ ID NOS. 13, 14), C6L (e.g., SEQ IDNOS. 15, 16), C7L (e.g., SEQ ID NOS. 17, 18), N1L (SEQ ID NOS. 19, 20),N2L (e.g., SEQ ID NOS. 21, 22), M1L (e.g., SEQ ID NOS. 23, 24), M2L(e.g., SEQ ID NOS. 25, 26), and K1L (e.g., SEQ ID NOS. 27, 28)) intoNYVAC (NYVAC+12) or NYVAC-C(NYVAC-C+12). The NYVAC-KC, NYVAC-C-KC,NYVAC+12, and NYVAC-C+12 have other attenuating deletions (present inthe “wild-type” NYVAC and NYVAC-C vectors), and thus remain relativelyattenuated despite being replication competent.

To produce NYVAC-KC, the C7L and K1L genes from the Copenhagen strain ofvaccinia virus were inserted back into the genome of either NYVAC orNYVAC-C. Each gene, plus a corresponding portion of the flankingregions, was amplified by PCR. The two fragments were combined into onefragment using PCR. The entire cassette containing both genes andflanking regions homologous to the adjacent genes of the NYVAC genomewas inserted into NYVAC-C by in vivo recombination. Recombinants wereselected by growth on RK-13 cells. The resulting viruses are called“NYVAC-KC” and “NYVAC-C-KC”, respectively (FIG. 9).

To produce the recombinant vector “NYVAC+12” and “NYVAC-C+12”, C1L(e.g., SEQ ID NOS. 5, 6), C2L (e.g., SEQ ID NOS. 7, 8), C3L (e.g., SEQID NOS. 9, 10), C4L (SEQ ID NOS. 11, 12), C5L (e.g., SEQ ID NOS. 13,14), C6L (e.g., SEQ ID NOS. 15, 16), C7L (e.g., SEQ ID NOS. 17, 18), N1L(SEQ ID NOS. 19, 20), N2L (e.g., SEQ ID NOS. 21, 22), M1L (e.g., SEQ IDNOS. 23, 24), M2L (e.g., SEQ ID NOS. 25, 26), and K1L (e.g., SEQ ID NOS.27, 28), which span the region from C7L to K1L of the Copenhagen strainof vaccinia virus (FIG. 2) were inserted (e.g., incorporated) back intogenome of either NYVAC or NYVAC-C, respectively (FIG. 10). The entirecassette of genes, with sequences flanking K1L and C7L, was prepared bylong-range PCR. The entire cassette was inserted into NYVAC or NYVAC-Cby in vivo recombination. Recombinants were selected by growth on RK-13cells.

The K1L (e.g, SEQ ID NO. 28 encoding SEQ ID NO. 27) and C7L (e.g, SEQ IDNO. 18 encoding SEQ ID NO. 17) genes of VACV Copenhagen were re-insertedinto NYVAC-ΔB8R/ΔB19R and NYVAC-C-ΔB8R/ΔB19R, as described above, toyield the viruses “NYVAC-KC-ΔB8R/ΔB19R” and “NYVAC-C-KC-ΔB8R/ΔB19R”,respectively. RK-13 cells were co-infected with “NYVAC-C+12-ATVh” (seebelow) and NYVAC-C-ΔB8R/ΔB19R, or the corresponding viruses lacking HIVgenes (e.g., NYVAC-KC-ΔB8R/ΔB19R and NYVAC+12-ATVh), to screen forrecombinant viruses containing an intact host range region. Individualplaques were screened by PCR to identify recombinants containing ATV andlacking B8R and B19R.

2. NYVAC-C-KC-ATVh and NYVAC-C+12-ATVh Recombinant Vectors

Double-stranded RNA (dsRNA) is a potent inducer of signalling throughstress related signalling pathways, such as TRL3/RIG1 and the p38 MAPkinase pathway. Signalling through these pathways leads to activation ofpro-inflammatory transcription factors ATF-2, NF-κB and IRF-3. Vacciniavirus blocks signalling by dsRNA by encoding a dsRNA-binding protein(the product of the E3L gene) that sequesters dsRNA and preventssignalling leading to activation of the pro-inflammatory transcriptionfactors IRF-3, NF-κB, and ATF-2. Vaccinia virus lacking E3L (VVΔF3L)induces signalling that leads to activation of these threepro-inflammatory transcription factors. This induces pro-inflammatorygene expression and induces a potent Th1 dominated immune response inmice, despite replicating to three logs lower titer than wild-typevaccinia (wtVV). However, utility of VVΔE3L is limited by activation ofRNA-dependent protein kinase (PKR) by dsRNA in infected cells.Activation of PKR in cells infected with VVΔE3L leads to a rapidinhibition of viral protein synthesis, limiting gene expression to thefirst four hours of infection.

To overcome this deficit, recombinant vectors have been developed thatreplace the E3L gene in vaccinia with the ATV eIF2αH (SEQ ID NO. 29, 30)which is known to be a potent, non-dsRNA-binding inhibitor of PKR. TheeIF2αH gene from ATV (Accession No. EU51233.1) (e.g., SEQ ID NO. 30) wascloned between the BamH1 and BclI sites in the pre-existing transferplasmid pMPE3ΔGPTMCS (Kibler et al. 1997. Double-stranded RNA is atrigger for apoptosis in vaccinia virus-infected cells. J Virol71:1992-2003). A map of the resulting plasmid, called pMPATVhom, isshown in FIG. 11. Transient dominant selection (mycophenolic acidresistance) was used to replace the E3L gene in NYVAC+12 and NYVAC-C+12with the ATV eIF2αH gene (producing “NYVAC+12-ATVh” and“NYVAC-C+12-ATVh”). Correct insertion was confirmed by PCR.

It is believed that virus having the E3L gene replaced by the ATV eIF2αHgene induces signal transduction through NF-KB and IRF-3, while sparingviral protein synthesis from the inhibitory effects of PKR activation.Replication of viruses expressing ATV eIF2αH was limited to a singleround by replacing the E3L gene with the gene for the ATV PKR inhibitor.Unlike its parental virus, this virus is highly sensitive to anti-viraleffects of interferon. Without being bound by theory, it is believedthat the unique interferon-sensitivity of this virus limits replicationto a single round. In addition to limiting replication to a single roundin human cells, this modification provides increased pro-inflammatorysignal transduction and increased pro-inflammatory gene expression tooccur in infected cells. This virus also induces a potent Th1 dominatedimmune response at low doses. Thus, this virus has the intrinsic safetyprofile of NYVAC with replication limited by induction and sensitivityto IFN, and increased pro-inflammatory signal transduction and increasedpro-inflammatory gene expression.

C. Recombinant Vector Compositions

For administration to a host, recombinant virus is typically, but notnecessarily (e.g., for the animal experiments described herein)maintained in a liquid form, with an extractable volume of 1ml to 1.1 mlin single dose 3 ml vials stored at −20° C. The composition containsapproximately 10⁸ DICC₅₀ recombinant vector (e.g recombinant NYVAC),0.25 ml 10 mM Tris-HCl buffer; pH 7.5, 0.25 ml Virus stabilizer(lactoglutamate); and, 0.5 ml freeze-drying medium.

D. Immunological Data

The recombinant viruses generated herein were tested for their abilityto improve the immune response. The results of these studies are shownbelow.

1. T-Cell Assays

Analysis of the in vitro immunogenicity of the replication-competentNYVAC-C—KC and NYVAC-C+12-ATVh revealed a significant improvement of theability to stimulate recall HIV-1-specific CD8 T-cell proliferativeresponses as compared to non-replication competent NYVAC-C(e.g.,“wild-type” NYVAC-C). All of the replication competent recombinantviruses (e.g., NYVAC-C—KC and NYVAC-C+12-ATVh) tested were able toinduce an HIV-1-specific CD8 T-cell responses in the range of 10-15%,whereas NYVAC-C exhibited a range of CD8 T-cell responses below 0.5%.These data were obtained in several independent experiments; twoexamples are shown in FIG. 12A. Furthermore, deletion of B8R and B19Rgenes on NYVAC-C-KC (e.g., NYVAC-C-KC-ΔB8R/ΔB19R) further increased thein vitro immunogenicity in the range of about 30%, as shown in FIG. 12B.Of note, these viruses were tested in a dose-dependent manner (rangingfrom 10⁷-10³ PFU, i.e. corresponding to a range of MOI going from10-0.001) in a conventional 6-day CFSE proliferation assay. Theproportion of proliferating cells (i.e. CFSE^(low) cells) was gated onlive CD3⁺CD8⁺ T cells after 6 days of in vitro stimulation with thedifferent doses of virus.

2. DC Maturation and Cross Presentation

The above-described NYVAC recombinant vectors were tested for theireffect on dendritic cell (DC) maturation and antigen processing to HIVspecific CD8 T cells. Maturation of monocyte derived human DCs wasmeasured 48 hrs post infection by the expression levels of severalco-stimulatory molecules by FACS analysis. Antigen presentation wasanalyzed after direct infection of DCs and after cross presentation,where the DCs have been incubated with infected apoptotic HeLa cells.Cytokine production by HIV-specific CD8 T cells was measured uponovernight stimulation with the DCs, either directly infected orincubated with HeLa cells.

Enhanced DC maturation was repeatedly observed in DCs infected with theB19R single and B8R/B19R double deletion mutants. Results from arepresentative experiment are shown in FIG. 13A. Forty eight hours afterinfection, increased expression of CD86, HLA-DR, HLA-A2 and CD80 wasobserved. These and other single deletion mutants, however, are notdifferent from NYVAC-C in their antigen presentation. In contrast, thereplication competent virus variants showed enhanced antigenpresentation in both direct and cross presentation assays compared toNYVAC-C, as determined by the number of single, double and triplecytokine (IFN-γ, TNF-α, MIP-1β) producing CD8 T cells. Results from arepresentative experiment of NYVAC-C KC are shown in FIG. 13B.

3. Macrophages

The innate immune response elicited by wild-type and modified NYVACpoxvirus was assessed by measuring IL-8 production by human THP-1macrophages (FIG. 14A) and whole blood (FIG. 14B). THP-1 cells (TIB-202,American Type Culture Collection) were differentiated into macrophagesby treatment with 0.5 μM phorbol 12-myristate 13-acetate for 24 h. THP-1cells were then infected with increasing multiplicity of infection ofwild-type and mutant NYVAC and NYVAC-C. After 1 h of contact with cells,the virus inoculum was removed and fresh medium added to the cultures.Cell-culture supernatants collected 24 h after infection were used toquantify IL-8 by ELISA. For whole blood assay, 100 μl of heparinizedwhole blood collected from three healthy volunteers were diluted 5-foldin RPMI 1640 medium containing viruses and incubated for 24 h at 37° C.in the presence of 5% CO₂. Samples were centrifuged, and cell-freesupernatants collected to quantify IL-8 and TNF by ELISA.

Results showed that expression of the clade-C gag-pol-nef HIVpolypeptides in NYVAC (ie NYVAC-C) enhanced IL-8 production by THP-1cells by two to four-fold (FIG. 14A). NYVAC-C with B8R, B19R andB8R-B19R gene deletions and replication competent NYVAC-KC andNYVAC-C-KC induced more IL-8 than NYVAC, but did not increase the IL-8response as compared to NYVAC-C. In whole blood, NYVAC-C induced lessIL-8 than NYVAC, whereas NYVAC-C with B19R and B8R-B19R gene deletionsand replication competent NYVAC-KC, NYVAC-C-KC, NYVAC-C+12 andNYVAC-C+12-ATV increased IL-8 release by 1.5 to 2.5-fold. NYVAC-C,NYVAC-C with B19R and B8R-B19R gene deletions and replication competentNYVAC-KC and NYVAC-C+12-ATV also increased TNF release by whole blood.

4. Gene Array

Gene expression profiling was conducted following PD-1 engagement.Myeloid or plasmacytoid dendritic cells were infected with wild type andmutant pox viruses for 6 hours. Cells were harvested and RNA wasextracted using Qiagen RNeasy™ kit (Cat #74104) according to themanufacturer's instructions. DNA was then hybridized on Illumina™ chips.Quantification was done using Illumina BeadStation™ 500GX scanner andIllumina BeadStudio™ 3 Software. Illumina gene averaged data wasexported from BeadStudio™ as raw data and was screened for quality(visual inspection of the chip image, analysis of the Illumina controls,diagnostic plots). Outliers were removed before subsequent analysis. Thedata was normalized using quantile method. Genes having intensitiesbelow background across all samples were filtered out and values belowbackground were surrogate replaced. The data was log 2 transformedbefore its analysis in R statistical package “Linear models formicroarray analysis” (LIMMA) where a fold change greater or equal to1.5, or less or equal to −1.5 and a moderated p-value less or equal to0.05 was considered significant. The NYVAC-C-ΔB8R/ΔB19R double mutantsinduced gene expression profiles similar to those induces by MVAexpressing the C clade, as described below:

-   -   Enhanced expression of early and late chemokines (CXCl10,        CXCL13, CXCL9, CXCL16; FIG. 15A);    -   Enhanced expression of chemokines that attract T cells, B cells,        NK cells and neutrophils;    -   Enhanced expression of cytokines which activate T cells (IL-15)        (FIG. 15A);    -   Enhanced expression of the IFN-α and IFN-β “machinery” (FIG.        15B);    -   Enhanced expression pathogen sensory molecules including RIG-1,        TLR-7 (FIG. 15C);    -   Induced the expression of the inflammsomes genes (FIG. 15D).    -   Induced a unique transcriptional network including but not        limited to IRF-1, IRF-7, STAT-5, NFKB, STAT3, STAT1, and IRF-10;        and,    -   Induced a transcriptional network signature resembling that        induced by YF vaccine.        It is noted that similar gene expression signatures were        elicited by macrophages to and DC.

All documents cited in this disclosure are hereby incorporated into thisdisclosure in their entirety. While the present invention has beendescribed in terms of the preferred embodiments, it is understood thatvariations and modifications will occur to those skilled in the art.Therefore, it is intended that the appended claims cover all suchequivalent variations that come within the scope of the invention asclaimed.

1-34. (canceled)
 35. A recombinant NYVAC vector encoding at least onepolypeptide selected from the group consisting of C1L (SEQ ID NO. 5),C2L (SEQ ID NO. 7), C3L (SEQ ID NO. 9), C4L (SEQ ID NO. 11), C5L (SEQ IDNO. 13), C6L (SEQ ID NO. 15), C7L (SEQ ID NO. 17), N1L (SEQ ID NO. 19),N2L (SEQ ID NO. 21), M1L (SEQ ID NO. 23), M2L (SEQ ID NO. 25) and K1L(SEQ ID NO. 27).